阅读说明：本技术 一种基因编辑载体及其编辑基因的方法和应用 (Gene editing vector and method and application for editing gene ) 是由 许凯 张望 智海剑 于 2021-05-11 设计创作，主要内容包括：本发明涉及基因工程技术领域,尤其涉及一种基因编辑载体及其编辑基因的方法和应用；所述基因编辑载体包括顺序排列的马铃薯Y病毒属病毒从5’-UTR区至衣壳蛋白区的序列、gRNA、插入序列和3’-UTR区；所述插入序列包括：编码所述马铃薯Y病毒属病毒的病毒衣壳蛋白的核酸序列。本发明通过马铃薯Y病毒属病毒构建基因编辑系统,可以浸染现有技术涉及较少的大豆、马铃薯等多种作物,扩大了可基因编辑的对象和范围。同时,还解决了马铃薯Y病毒属的病毒由于不能产生亚基因组RNA,导致难以通过常规方式产生gRNA的技术问题,在基因工程技术领域具有重要意义。(The invention relates to the technical field of genetic engineering, in particular to a gene editing vector and a method and application for editing genes thereof; the gene editing vector comprises a sequence from a 5 '-UTR region to a capsid protein region of the potyvirus, a gRNA, an insertion sequence and a 3' -UTR region which are sequentially arranged; the insertion sequence comprises: a nucleic acid sequence encoding a viral capsid protein of said potyvirus. The gene editing system is constructed by the potyvirus, so that a plurality of crops such as soybeans, potatoes and the like in the prior art can be infected, and the object and range of gene editing can be enlarged. Meanwhile, the technical problem that the gRNA is difficult to generate in a conventional mode due to the fact that the virus of potyvirus cannot generate subgenomic RNA is solved, and the method has important significance in the technical field of genetic engineering.)

1. A gene editing vector comprising: a sequence of the potyvirus from the 5 '-UTR region to the capsid protein region, a gRNA, an insertion sequence and a 3' -UTR region in sequence;

the insertion sequence is optionally any one of:

i) a nucleic acid sequence encoding a viral capsid protein of said potyvirus;

ii) the nucleic acid sequence shown in i) can still form a nucleic acid sequence of a complete RNA element required for virus replication together with the nucleic acid sequence of the 3' -UTR region after one or more nucleotide substitutions, deletions and/or additions.

2. A gene editing vector according to claim 1, wherein the sequence of the capsid protein region is codon optimized for changes in its nucleic acid sequence;

preferably, the optimized sequence does not have a contiguous, fully identical homologous fragment of greater than 30 bases compared to the sequence before optimization.

3. A gene editing vector according to claim 1 or 2, wherein when the insertion sequence is selected from ii), the insertion sequence is obtained by the following process:

dividing the nucleic acid sequence encoding the virus capsid protein of the potyvirus into a plurality of segments by 100 nt-300 nt, randomly removing at least one segment of the nucleic acid sequence, and evaluating the virus replication capacity and/or gene editing efficiency of the remaining nucleic acid sequence in the vector; if replication and gene editing of the potyvirus can be smoothly performed, the remaining nucleic acid sequence is the insertion sequence.

4. A gene editing vector according to any one of claims 1-3, characterized in that between the sequence of the capsid protein region and the gRNA, a nucleic acid sequence that can self-cleave or be cleaved by an intracellular endonuclease, preferably a nucleic acid sequence encoding a ribozyme or Pre-tRNA, is further included.

5. The gene editing vector of claim 4, wherein the ribozyme is a mutant cylindrical ribozyme; preferably, the nucleotide sequence of the mutant type columnar ribozyme is shown as SEQ ID NO. 2.

6. The gene-editing vector of claim 1, wherein the potyvirus virus comprises potyvirus (Potato virus Y), Soybean mosaic virus (Soybean mosaic virus), Bean common mosaic virus (Bean common mosaic virus), Bean yellow mosaic virus (Bean yellow mosaic virus), black eye Cowpea mosaic virus (black eye Cowpea mosaic virus), Bean aphid mosaic virus (Cowpea aphid-borne mosaic virus), Cowpea green vein banding virus (Cowpea green mosaic virus), Guar agapanthus virus (Guar gum thomless virus), mung Bean mosaic virus (mung Bean mosaic virus), Pea seed mosaic virus (Pea mosaic virus), Pea mosaic virus (Pea mosaic virus), Peanut mosaic virus (Peanut mosaic virus), Peanut mosaic virus (Maize mosaic virus), and Peanut mosaic virus (Maize yellow mosaic virus), Watermelon mosaic virus (watermelonlon mosaic virus), Sugarcane mosaic virus (sugar cane mosaic virus), Wheat streak mosaic virus (Wheat streak mosaic virus), Turnip mosaic virus (Turnip mosaic virus), Plum pox virus (Plum pox virus), Sunflower mosaic virus (Sunflower mosaic virus).

7. The gene editing vector of claim 1, wherein the gene editing vector comprises a nucleotide sequence set forth as SEQ ID No.3 or SEQ ID No. 4.

8. A method of gene editing, comprising: performing gene editing on a target plant by using the gene editing vector of any one of claims 1 to 7;

the target plant comprises one or more of soybean, kidney bean, cowpea, mung bean, tobacco, watermelon, potato, alfalfa, sweet potato, corn, wheat or barley.

9. The gene editing method according to claim 8, comprising:

transforming the gene editing vector into the target plant by an agrobacterium infiltration method, a gene gun bombardment method or a friction inoculation method.

10. Use of the gene editing vector of any one of claims 1 to 7, or the gene editing method of claim 8 or 9, for modifying a plant phenotype; the plant phenotypes include: yield, disease resistance, stress resistance, seed quality and fruit quality.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene editing vector, a method for editing a gene thereof and application thereof.

Background

In many bacteria and most archaea, there is a defense system available against foreign DNA (e.g. phage), called CRISPR/Cas9(Clustered regulated shorten coiled materials/CRISPR-associated Cas9 end effector) system (Bhaya et al, 2011). The CRISPR/Cas9 system comprises two basic components, one is the endonuclease Cas9 protein and the other is the grna (guide rna). Under certain conditions, the gRNA can form a complex with the Cas9 protein, and through base complementary pairing, the gRNA can guide the Cas9 protein to the target DNA, and then the Cas9 protein can bind and cleave the target double-stranded DNA, thereby causing a target DNA double-strand break (double strand break). DNA repair mechanisms within cells repair damaged double-stranded DNA, including NHEJ (non-homologus end-joining) and HDR (homology-directed repair) (Hsu et al, 2014). Most of them play a major role in NHEJ, but NHEJ repair mechanism mediated repair of double-stranded DNA is often inaccurate, and different types of mutations, such as base insertion, base deletion and base substitution, may be introduced into the repaired DNA double-strand, which may further cause the change or deletion of target DNA function, thereby achieving the purpose of "gene editing". With the CRISPR/Cas9 system, people realize the editing of genomes of various organisms including animals, plants and microorganisms, thereby realizing the targeted modification of certain characters of target organisms. For example, a transient expression vector containing CRISPR/Cas 9-related components is introduced into maize embryos by a gene gun, and the thermo-sensitive genetic male-sterility 5(TMS5) gene of maize is edited, so that maize with male sterility can be obtained at 32 ℃ (Li et al, 2017). However, such a strategy for gene editing by using the CRISPR/Cas9 system is based on a transient expression vector, and a plasmid capable of expressing the CRISPR/Cas9 component needs to be introduced into a target tissue or cell by means of a special external device (such as a gene gun). Based on such methods, the number of cells that can obtain the CRISPR/Cas9 component is limited, and the amount of CRISPR/Cas9 component that enters the target cell is also limited. To obtain the mutant of interest, it is often necessary to invest a lot of time and labor for the relevant operations, such as obtaining immature embryos of plants and performing tissue culture.

In recent years, in order to facilitate the introduction of CRISPR/Cas9 components into target tissues and cells, simplify the operation procedure, and improve the efficiency of gene editing, reports have been made in which grnas are introduced into target tissues and expressed in large quantities using plant viral vectors to achieve the purpose of plant gene editing, for example, host plant genomes are edited using geminivirus, Tobacco Rattle Virus (TRV), Tobacco Mosaic Virus (TMV), and Barley Streak Mosaic Virus (BSMV).

Taking a geminivirus Wheat Dwarf Virus (WDV) as an example, WDV is a plant DNA virus, and authors use the modified WDV to express a large amount of Cas9 protein and a DNA template for HDR repair to achieve gene targeting of endogenous genes. Thanks to the efficient replication of the engineered WDV replicon in the target cells, its editing efficiency on the target gene is 12 times that of non-viral vectors (Gil-Humanes et al, 2017). Both the motor and capsid proteins of the engineered WDV virus are removed, losing the ability to move, but are able to replicate efficiently. Since WDV is DNA virus, the authors express donor DNA in large quantities by using WDV replicons and use the donor DNA as a template for homologous recombination and repair to achieve gene targeting of plant endogenous genes. The gene editing vector based on the WDV replicon can simultaneously express Cas9 and gRNA in large quantity, and can edit endogenous genes of crops such as corn, wheat, rice and the like.

However, the plant geminivirus is a DNA virus, and when infecting plants, the geminivirus may compete with host cells for relevant factors such as replication and translation, interfere normal growth of the plants, and bring difficulty to tissue culture regeneration of the plants. Geminivirus replicon-based gene editing vectors are often unable to move and spread in plants. In addition, geminivirus is still one of the most serious types of virus threatening crops at present, can cause severe symptoms and cause huge damage to crops, and can be transmitted by insects (such as bemisia tabaci) and, once spread, is difficult to control.

Tobacco Rattle Virus (TRV) is a positive-sense single-stranded RNA virus whose genome comprises two RNA strands. By modifying TRV RNA2, the authors successfully utilized TRV to express gRNA in Nicotiana benthamiana and cooperate with Cas9 protein to achieve editing of endogenous genes of Nicotiana benthamiana (Ali et al, 2015). The authors disrupted the 2b protein (nematode transmission-associated) and 2c protein (function unknown) expression cassettes on TRV RNA2, and this alteration did not affect the systemic movement of the virus on burley tobacco. Transcription was driven by cloning the gRNA downstream of the CP protein expression cassette on TRV RNA2, and by the homologous Pea Early Browning Virus (PEBV) promoter (192 bp). By applying the strategy, the PDS and PCNA genes of the injection leaf and the system leaf of the Nicotiana benthamiana are successfully edited, and the editing efficiency is about 50%.

However, the genome of TRV contains two single-stranded RNAs, and although the TRV-based gene editing vector maintains the ability of systemic movement, the TRV has a limited host range and is generally not infected with monocotyledons including important crops such as barley, wheat, and corn, and is not infected with dicotyledons such as soybean, thereby limiting its application to these important crops.

In 2017, researchers reported that Tobacco Mosaic Virus (TMV) was used to edit endogenous genes of nicotiana benthamiana (Cody et al, 2017). TMV is also a single-stranded RNA virus, and the author deletes the CP protein expression cassette of TMV, and the TMV mutant loses the ability of systemic movement in Nicotiana benthamiana, but still retains the replication ability, and can replicate on the Agrobacterium-infiltrated leaves of Nicotiana benthamiana by an Agrobacterium-infiltrated method. The authors cloned the gRNA into the MP protein expression cassette and transcribed the gRNA from the subgenomic promoter of the CP. There are about 60nt extra bases between the transcription start site of the subgenomic promoter of CP and gRNA, and there is a UTR sequence of nearly 200bp behind gRNA, in this case, the authors found that the TMV-based gene editing vector can still edit the endogenous gene AGO1 of Nicotiana benthamiana, and the efficiency can reach 70%. Furthermore, the authors found that multiple grnas could be directly concatenated and that they all could function.

However, when it is designed, the movement protein of the virus itself is removed, so that the virus loses the ability of system movement, and the site where it acts is limited. Furthermore, TMV also cannot infect gramineous crops such as wheat, maize, etc., nor be transmitted through seeds, i.e., has the potential to edit T0 seed directly and harvest target mutants quickly.

Barley Mosaic Virus (BSMV) belongs to the plant Baculoviridae, the genus Barley Virus, and its genome contains three positive-sense single-stranded RNAs, respectively designated RNA α, RNA β and RNA γ. It is known that there are multiple sites on barley streak mosaic virus genome into which foreign fragments can be inserted without affecting replication and movement of the virus itself, such as 5 'and 3' ends of γ b; the 3' end of TGB 3; furthermore, the authors found that the middle part of the CP sequence of barley streak mosaic virus could be replaced with an exogenous fragment without affecting the movement of the virus (Hu, Jiancheng, Li, et al.,2019, Molecular Plant Pathology), and that these positions could be used for insertion and expression of gRNAs. The 3' end of one of the sites- γ b, preferred by the authors, was used for insertion and expression of grnas. The gRNA comprises two portions, a spacer portion at the 5 'end and an immediately contiguous backbone portion at the 3' end; the nucleotide sequence of the spacer can be changed, and the length of the spacer can also be changed according to the difference of the Cas9 protein. The upstream and/or downstream backbone portion of the Spacer may have additional sequences in addition to the sequences of the barley mosaic virus genome itself, may be sequences of one or more grnas and/or may have sequences other than the virus itself and the grnas. The gene editing vector based on BSMV can express gRNA in large quantity and successfully edit endogenous genes of Nicotiana benthamiana, wheat and corn.

In 2020, a gene editing system using a plant negative strand RNA virus (Sonchus yellow net rhabdovirus, SYNV) was reported (Xiaonan Ma, et al, 2020, Nature Plants), in which a gRNA was placed in the middle of an N/P gene in a virus positive strand RNA and a sequence existing in the middle of the N/P gene was added to control its transcription, and pre-tRNA sequences were added to 5 'and 3' ends of the gRNA to help the gRNA to be cleaved from mRNA transcribed by the virus. In the system, a gene for expressing Cas9 protein is added at the downstream of the gRNA, so that the virus can express not only the gRNA but also the Cas9 protein, and the gene editing independent of DNA and only dependent on RNA (virus) is realized. However, due to the host range limitation of SYNV, this system has only proven effective in Nicotiana benthamiana.

In 2020, it was reported that a modified Tobacco Rattle Virus (TRV) gene editing system could be introduced into seeds for editing (heritable) (Evan e.ellison, et al, 2020, Nature Plants). This system was based on the TRV system reported in 2015, which used the RNA2 of TRV to remove the 2b and 2c expression cassettes, but added the Pea Early Browning Virus (PEBV) promoter subgenomic RNA promoter (192bp) of the same genus to drive the transcription of gRNA. However, unlike the report in 2015, this vector added the complete gene of Flowering gene Flowering LocusT (FT), or mFT gene with mutant initiation codon, or truncated mutant downstream of gRNA. Since fragments of the flowering gene can move systemically in the plant to the growing point, gRNA delivery into seeds is facilitated, increasing editing efficiency in seeds. However, the host range of TRV is limited, and the TRV cannot infect important crops such as barley, wheat, corn and the like.

In summary, current gene editing systems using plant viruses to deliver guide RNA rely mostly on Cas9 transgenic plants (except SYNV), and the host range is still not broad enough, and the available hosts are still limited.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a gene editing vector, a gene editing method and application, and particularly relates to a gene editing system constructed by potyviruses with extremely wide host range and the same genome structure, so that the object and range of gene editing are widened.

In a first aspect, the present invention provides a gene editing vector comprising: a sequence of the potyvirus from the 5 '-UTR region to the capsid protein region, a gRNA, an insertion sequence and a 3' -UTR region in sequence;

the insertion sequence is optionally any one of:

i) a nucleic acid sequence encoding a viral capsid protein of said potyvirus;

ii) the nucleic acid sequence shown in i) can still form a nucleic acid sequence of a complete RNA element required for virus replication together with the nucleic acid sequence of the 3' -UTR region after one or more nucleotide substitutions, deletions and/or additions.

The gRNA, the insertion sequence and the 3' -UTR region are added after the stop codon of the capsid protein of the potyvirus virus. Wherein the insertion sequence and the 3' -UTR region together form the complete RNA element (or promoter sequence) required for viral replication, enabling a virus carrying a gRNA to replicate normally and systemically infect a host plant, thereby performing gene editing on the infected tissue.

Further, the sequence of the capsid protein region is codon optimized to increase the free energy of the secondary structure of the nucleic acid sequence;

preferably, the optimized sequence does not have a contiguous, fully identical homologous fragment of greater than 30 bases compared to the sequence before optimization.

The inventors performed codon optimization of the nucleic acid sequence encoding the viral capsid protein in potyviruses with the aim of avoiding the consensus with the newly inserted viral capsid protein sequence after the stop codon to generate virus-mediated homologous recombination, thereby allowing the gRNA to be lost in the middle of the two sequences. However, it is possible that non-optimized viral capsid proteins may also play a role, as judged by routine knowledge in the art.

Further, the insertion sequence is processed by the following scheme i) or scheme ii):

i) selecting a nucleic acid sequence encoding a viral capsid protein of the potyvirus as an insertion sequence;

ii) dividing the nucleic acid sequence encoding the viral capsid protein of the potyvirus into a plurality of fragments of 100 nt and 300nt, optionally removing at least one of the nucleic acid sequence fragments, and evaluating the remaining nucleic acid sequence in the vector for viral replication ability and/or gene editing efficiency; if replication and gene editing of the potyvirus can be smoothly performed, the remaining nucleic acid sequence is the insertion sequence.

Specifically, the inventors truncated the complete nucleic acid sequence encoding the viral capsid protein from the 5 'end step by step (each truncation is basically in the unit of 100-300 nt) according to the distribution characteristics of the neck-loop structure on the secondary structure by analyzing the secondary structure of the viral capsid protein CP and the sequence of the 3' -UTR together, without destroying the neck-loop structure, and evaluated and screened according to the replication ability and/or the gene editing efficiency of the virus; if the replication of the potyvirus virus can be smoothly performed and gene editing can be performed, the remaining nucleic acid sequence is the insertion sequence. In addition, the inventors have found that by this regular truncation of the sequences, some are more efficient in editing than the complete viral capsid protein sequence.

Further, a nucleic acid sequence that can self-cleave or be cleaved by an intracellular endonuclease, preferably a nucleic acid sequence encoding a ribozyme or a Pre-tRNA, is also included between the sequence of the capsid protein region and the gRNA.

Further, the ribozyme is a mutant type columnar ribozyme; preferably, the nucleotide sequence of the mutant type pillared ribozyme comprises the nucleotide sequence shown as SEQ ID NO. 2.

The preferred inserted ribozymes herein of the present invention are mutant cylindrical ribozymes (Hammer head ribozymes), although other similar ribozymes may be used to release grnas in the prior art.

Further, the potyvirus includes, but is not limited to, potyvirus (potatovirus Y), Soybean mosaic virus (Soybean mosaic virus), Bean common mosaic virus (Bean common mosaic virus), Bean yellow mosaic virus (Bean yellow mosaic virus), black-eyed Cowpea mosaic virus (black Cowpea mosaic virus), Cowpea aphid mosaic virus (Cowpea aphid-borne mosaic virus), Cowpea green vein banding virus (Cowpea green mosaic virus), Guar asymptomatic virus (Guar mosaic virus), mung Bean mosaic virus (mung Bean mosaic virus), Pea seed mosaic virus (Pea mosaic virus), Peanut mottle virus (Peanut mottle virus), Peanut mosaic virus (Watermelon mosaic virus), Watermelon mosaic virus (Watermelon mosaic virus), and Watermelon mosaic virus (Watermelon mosaic virus), Pea mosaic virus (Watermelon mosaic virus), and Bean mosaic virus (Watermelon mosaic virus) and corn mosaic virus (Watermelon mosaic virus), Sugarcane mosaic virus (Sugarcane mosaic virus), Wheat streak mosaic virus (Wheat streak mosaic virus), Turnip mosaic virus (Turnip mosaic virus), Plum pox virus (Plum pox virus), or Sunflower mosaic virus (Sun flower mosaic virus).

Viruses of the genus potyvirus have the same genome structure (as shown in a in fig. 3), and can be theoretically applied to the design concept of the gene editing vector provided by the present invention.

The design concept of the gene editing vector can be applied to all potyviruses.

Further, the gene editing vector comprises a nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO. 4.

In a second aspect, the present invention provides a gene editing method for gene editing of a target plant using the gene editing vector.

Further, the gene editing vector is transformed into the target plant by agroinfiltration, particle gun bombardment or friction inoculation.

Further, the target plant is a host plant capable of being infected by potyvirus, comprising:

one or more of soybean, kidney bean, cowpea, mung bean, tobacco, watermelon, potato, alfalfa, sweet potato, corn, wheat or barley.

The invention further provides the use of the gene editing vector, or the method of gene editing, for modifying a plant phenotype; the plant phenotype preferably comprises: yield, disease resistance, stress resistance, seed quality and fruit quality.

The gene editing vector provided by the invention can be introduced into plant tissue cells by methods such as gene gun bombardment, agrobacterium infiltration, friction inoculation and the like, and can be copied and moved in Nicotiana benthamiana; the gene editing vector contains a gRNA sequence, and can realize the editing of plant genomes such as Nicotiana benthamiana under the condition of transient or transgenic expression of Cas protein. For example, the soybean mosaic virus-based gene editing vector can be inoculated on Nicotiana benthamiana by an agrobacterium infiltration method, and gene editing can be performed on plant tissues within a direct inoculation area and infected by a virus system under the condition of existence of spCas9 protein.

The invention has the following beneficial effects:

compared with the existing TRV-based gene editing vector, the gene editing vector provided by the invention can infect some important crops, including soybean, potato, alfalfa and the like;

compared with a TMV-based gene editing vector, the gene editing vector provided by the invention retains the system movement capability and replication capability of the virus in a host plant, so that the gene editing of plant tissue cells in a wider range can be realized, and the gene editing vector is not limited in the field of inoculation of the virus on plants;

compared with a gene editing vector based on geminivirus, the gene editing vector provided by the invention does not compete with plant cells for original parts such as DNA replication and interfere with the normal plant cell cycle on the premise of realizing higher editing efficiency; in addition, potyvirus, as an RNA virus, does not integrate into the genome of the plant and introduce additional foreign fragments into the plant genome;

compared with the gene editing vector based on BSMV, the virus of potyvirus has wide host range and potential for editing wider host plants, including soybean (soybean), bean (bean), cowpea (cowpea), mung bean (mung bean), watermelon (watermelonon), alfalfa (alfalfalfa), sweet potato (sweet potato) and the like.

The gene editing vector provided by the invention can insert a ribozyme sequence, a gRNA sequence and a complete or partial nucleic acid sequence for coding the virus capsid protein into a stop codon of a nucleic acid sequence of the virus capsid protein without influencing the system infection of the potyvirus virus on the Nicotiana benthamiana of a host plant; additional nucleotide sequences can be added at two ends of the gRNA sequence without damaging the normal function of the gRNA sequence; meanwhile, since the virus can move in the plant, the gene editing vector can edit the plant tissues outside the inoculation area.

The invention provides a gene editing vector which can realize the editing efficiency of more than 50% on the inoculated leaves of the Nicotiana benthamiana mGFP5 gene of 16c transgenic Nicotiana benthamiana which transiently expresses Cas9 or the PDS gene of the Cas9 transgenic Nicotiana benthamiana by PCR/RE experiments and calculation by adopting quality one software on the 6 th day of inoculation under the condition of transient or transgenic expression of Cas 9; on the 12 th day of inoculation, the mGFP5 gene of 16c transgenic Nicotiana benthamiana which transiently expresses Cas9 or the PDS gene of Cas9 transgenic Nicotiana benthamiana can achieve editing efficiency of more than 50% on the upper leaves of Nicotiana benthamiana through PCR/RE experiments and calculation by adopting quality one software.

Drawings

FIG. 1 is a schematic diagram showing the construction of a soybean mosaic virus-based gene editing vector provided in example 1 of the present invention.

FIG. 2 is a schematic diagram of the secondary structure of the RNA of the original capsid protein sequence plus 3' UTR sequence of soybean mosaic virus analyzed by mfold according to example 1 of the present invention; wherein the arrow is the truncated position.

FIG. 3 is a schematic diagram showing the structural comparison of potyvirus genomes before and after modification provided in example 1 of the present invention; wherein A is a structural schematic diagram of a potyvirus genome; b is a structural schematic diagram of a modified potyvirus genome for gene editing; c is a schematic diagram of the soybean mosaic virus gene structure for gene editing and a primer required for constructing the clone; d is a structural schematic diagram of the potato virus Y gene for gene editing and a primer required for constructing the clone; wherein CP is a sequence of a capsid protein region of a codon-optimized potyvirus, Rz denotes a ribozyme or other nucleic acid sequence having a self-cleaving function, target sequence and spCas9-gRNA scaffold denote a targeting sequence and a backbone sequence constituting a gRNA, CP sequence denotes an insertion sequence (a nucleic acid sequence of a full-length or truncated viral capsid protein), original 3 '-UTR denotes a 3' UTR sequence of a potyvirus before optimization; rz, gRNA, the insertion sequence and original 3 ' -UTR together constitute the modified virus 3 ' UTR sequence (New 3 ' -UTR).

FIG. 4 is a schematic diagram showing the result of Western blotting detecting the accumulation of CP of soybean mosaic virus with different versions inserted into gRNA and the accumulation of CP of wild-type soybean mosaic virus injected on day 6, which are provided by embodiment 1 of the present invention.

FIG. 5 is a schematic diagram showing the results of detecting gene editing efficiency of different versions of gRNA-inserted soybean mosaic virus on day 6 by agarose gel electrophoresis according to example 1 of the present invention; among them, since the NdeI cleavage site of the mGFP5 gene fragment was disrupted by gene editing, the edited gene fragment could not be cleaved with NdeI.

FIG. 6 shows the edited sequencing site of mGFP5 from leaf cells in the injection region of day 6 using ribozyme version 7 (SMV-HHRz-gRNA-insert #3) provided in example 1 of the present invention; wherein, CATATG is NdeI restriction enzyme cutting site, CGG is PAM; the horizontal bar represents the deletion mutation, and the bold represents the insertion mutation.

FIG. 7 shows the edited sequencing site of mGFP5 from leaf cells in the injection region of day 6 using ribozyme version 8 (SMV-HHRz-gRNA-insert #4) provided in example 1 of the present invention; wherein, CATATG is NdeI restriction enzyme cutting site, CGG is PAM; the horizontal bar represents the deletion mutation, and the bold represents the insertion mutation.

FIG. 8 is a schematic diagram showing the result of Western blotting detecting accumulation of viral CP in systemic leaves on day 12 of soybean mosaic virus with different versions of inserted gRNA and wild type soybean mosaic virus provided in example 1 of the present invention.

FIG. 9 is a graph showing the results of the agarose gel electrophoresis performed on the results of detecting the gene editing efficiency of soybean mosaic virus plus ribozyme in leaf cells systemically infected at day 12 in version 7 (SMV-HHRz-gRNA-insert #3) and version 8 (SMV-HHRz-gRNA-insert #4) according to example 1 of the present invention; among them, since the NdeI cleavage site of the mGFP5 gene fragment was disrupted by gene editing, the edited gene fragment could not be cleaved with NdeI.

FIG. 10 shows the mGFP5 editing site sequencing result of the soybean mosaic virus plus ribozyme version 7 (SMV-HHrz-gRNA-insertion sequence #3) system leaf cells on day 12 according to example 1 of the present invention; wherein, CATATG is NdeI restriction site, CGG is PAM, and the bar represents deletion mutation.

FIG. 11 shows the mGFP5 editing site sequencing result of the soybean mosaic virus plus ribozyme version 8 (SMV-HHrz-gRNA-insertion #4) system leaf cells on day 12 according to example 1 of the present invention; wherein, CATATG is NdeI restriction site, CGG is PAM, and the bar represents deletion mutation.

FIG. 12 is a schematic diagram showing the construction of a potato virus Y-based gene editing vector provided in example 2 of the present invention.

FIG. 13 is a schematic diagram showing the result of Western blotting detecting the accumulation amount of CP injected into the 6 th day by inserting the gRNA into the potato Yvirus and the wild-type potato Yvirus in example 2 of the present invention; in this case, the gene editing disrupted the NcoI cleavage site of the PDS gene fragment, and the gene fragment with the editing thus occurred was not cleaved with NcoI.

FIG. 14 is a diagram showing the results of agarose gel electrophoresis detecting gene editing efficiency of gRNA-inserted potato virus Y on day 6 in accordance with example 2 of the present invention.

FIG. 15 shows the sequencing results of the PDS editing site of the injection leaves on day 6 according to example 2 of the present invention; wherein CCATGG is NcoI enzyme cutting site, GGG is PAM, and the bar represents deletion mutation; bold represents insertional mutations and italics represent point mutations.

FIG. 16 is a schematic diagram showing the result of Western blotting detecting accumulation amounts of systemic leaf CP on day 12 of the gRNA-inserted potyvirus and wild-type potyvirus provided in example 2 of the present invention.

FIG. 17 is a schematic diagram showing the results of detecting the gene editing efficiency of the gRNA-inserted potato virus Y on day 12 by agarose gel electrophoresis in accordance with example 2 of the present invention; in this case, the gene editing disrupted the NcoI cleavage site of the PDS gene fragment, and the gene fragment with the editing thus occurred was not cleaved with NcoI.

FIG. 18 shows the sequencing results of the editing sites of the systematic leaf PDS on day 12 according to example 2 of the present invention; wherein CCATGG is NcoI enzyme cutting site, and GGG is PAM; horizontal bars represent deletion mutations, bold represents insertion mutations, and italics represents point mutations.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

In this embodiment, a CRISPR/Cas9 gene editing system using soybean mosaic virus as a vector is constructed, and the system is shown in fig. 1, and the construction process is as follows:

1. selecting a soybean mosaic virus SC7 isolate (Genbank Access #: MH919385) as a raw material;

2. a nucleic acid sequence encoding a capsid protein of a codon-optimized soybean mosaic virus SC7 isolate. Species selection common cigarette is optimized by one key by using an online website (https:// www.vectorbuilder.cn/tool/codon-optimization. html) to obtain an optimized nucleic acid sequence, and the optimized nucleic acid sequence of the soybean mosaic virus capsid protein is directly synthesized by genes and is used for replacing an original CP sequence in a reading frame as shown in SEQ ID NO. 1. (Note: codon optimization serves to avoid agreement with a newly inserted CP sequence after the stop codon to produce virus-mediated homologous recombination, thereby leaving the gRNA in the middle of the two sequences missing, but we still speculate that non-optimized CP may also play a role.)

3. The soybean mosaic virus is divided into 6 segments for PCR amplification, and simultaneously mutant hammerhead ribozymes, gRNAs (edited mGFP5, shown in SEQ ID NO. 48) and insertion sequences are integrated into the infectious clone of the soybean mosaic virus in a PCR mode, wherein the total number of the insertion sequences is 8 different versions. Wherein, the insertion sequence is based on the analysis of the secondary structure of the viral capsid protein CP and the sequence of the 3 '-UTR together (FIG. 2), the complete nucleic acid sequence encoding the viral capsid protein is truncated (in units of 100-300 nt) from the 5' end by the distribution characteristics of the neck-loop structures on the secondary structure without destroying the main independent neck-loop structures, and the truncated position is shown by an arrow in FIG. 2.

The overall concept of 8 different versions is shown as C in fig. 3, specifically, each different version is constructed as follows:

(1) versions 1-4 do not contain hammerhead ribozymes, but only grnas and insertion sequences # 1-4;

(2) versions 5-8 contain a hammerhead ribozyme, as well as the gRNA and insertion sequences # 1-4.

Where versions 1 and 5 contain insertion #1, versions 2 and 6 contain insertion #2, versions 3 and 7 contain insertion #3, and versions 4 and 8 contain insertion # 4. Wherein, the insertion sequence #1, the insertion sequence #2 and the insertion sequence #3 are truncated soybean mosaic virus capsid protein nucleic acid sequences, and the insertion sequence #4 is a full-length soybean mosaic virus capsid protein nucleic acid sequence.

The sequence of the inserted hammerhead ribozyme is shown as SEQ ID NO. 2. The nucleic acid sequence of the insertion sequence #1-4 is shown in SEQ ID NO.44-47, and the inserted gRNA framework sequence is shown in SEQ ID NO. 48.

The specific cloning steps are as follows:

(1) fragment 1 was amplified by primers SMV/PVY-frag1-F and SMV-frag1-R, fragment 2 by primers SMV-frag2-F and SMV-frag2-R, fragment 3 by primers SMV-frag3-F and SMV-frag3-R, intermediate product was obtained by amplification by primers SMV-frag4-F and SMV-frag4-R1, intermediate product was recovered as template after tapping, and fragment 4 was amplified by primers SMV-frag4-F and SMV-frag4-R2 using the complementary DNA (cDNA) of the soybean mosaic virus SC7 isolate as template.

(2) Using the optimized soybean mosaic virus capsid protein nucleic acid sequence of gene synthesis as a template, and amplifying a segment 5a by primers SMV-frag5-F, SMV-frag5a-R1, SMV-frag5a-R2 and SMV-frag5 a-R3; the soybean mosaic virus infectious DNA clone is used as a template, a fragment 6a is amplified by primers SMV-frag6a-F and SMV/PVY-frag6-R, a fragment 6b is amplified by primers SMV-frag6b-F and SMV/PVY-frag6-R, a fragment 6c is amplified by primers SMV-frag6c-F and SMV/PVY-frag6-R, and a fragment 6d is amplified by primers SMV-frag6d-F and SMV/PVY-frag 6-R.

(3) Fragment 5a was mixed with fragment 6a, 6b, 6c or 6d as a template, and amplified by primers SMV-frag5-F and SMV/PVY-frag6-R to give fragments 5a +6a, 5a +6b, 5a +6c, 5a +6d, respectively. The StuI and SmaI double-digested pCB301-314 and fragments 1,2,3 and 4 are mixed with fragments 5a +6a, 5a +6b, 5a +6C and 5a +6d, respectively, and clone versions 1 to 4 are obtained by yeast homologous recombination (see C in FIG. 3 for a cloning construction scheme).

(4) The optimized soybean mosaic virus capsid protein nucleic acid sequence synthesized by the gene is used as a template, and a fragment 5b is amplified by primers SMV-frag5-F, SMV-frag5b-R1, SMV-frag5b-R2, SMV-frag5b-R3, SMV-frag5a-R2 and SMV-frag5 a-R3.

(5) Fragment 5b was mixed with fragments 6a, 6b, 6c, 6d as templates, and amplified with primers SMV-frag5-F and SMV/PVY-frag6-R to give fragments 5b +6a, 5b +6b, 5b +6c, 5b +6d, respectively.

(6) The StuI and SmaI double-digested pCB301-314 and fragments 1,2,3 and 4 are mixed with fragments 5b +6a, 5b +6b, 5b +6C and 5b +6d, respectively, and clone versions 5 to 8 are obtained by yeast homologous recombination (see C in FIG. 3 for a cloning construction scheme).

4. The yeast plasmids of clone versions 1-8 are extracted in large quantity by the following specific method: the single colonies that grew out were picked up in 50ml SC-T-and shaken at 30 ℃ and 250rpm overnight. After centrifugation at 5000rpm for 5min, the supernatant was removed and 1.5ml sorbitol phosphate buffer (0.1M PBS containing 1.2M sorbitol, pH 7.4), 100ul helicase solution (0.12g/ml, helicase powder dissolved in a mixed solution containing 10% 0.1M PBS, 40% sterile water, 50% glycerol) and 10. mu.l RNaseA (10mg/ml) were added to 1g yeast pellet, vortexed and mixed, and then enzymolyzed overnight at 37 ℃. After the yeast cell wall is removed, the plasmid can be extracted by a conventional alkaline lysis method, and in the embodiment, a plasmid extraction kit (nuozan, Nanjing, China) is used.

5. With CaCl₂Preparing agrobacterium tumefaciens strain C58C1 competence by a treatment method, and then transforming yeast plasmid into newly prepared C58C1 competence by a liquid nitrogen freeze-thaw method, wherein the specific method comprises the following steps:

activated C58C1 was shaken overnight at 250rpm in 2ml LB (containing 50. mu.g/ml streptomycin and 100. mu.g/ml rifampicin) at 30 ℃. The 2ml of the suspension was added to 50ml of LB (containing no antibiotics) and shaken at 250rpm at 30 ℃ until OD was 0.5. Placing the bacterial liquid on ice for 10min, then removing supernatant at 5000rpm at 4 deg.C for 10min, and suspending the precipitate in 1ml of pre-cooled 20mM CaCl₂. 100 μ l of competence was added to a centrifuge tube to which yeast plasmids were added in advance, and the mixture was frozen on ice for 5min, frozen on liquid nitrogen for 5min, at 37 ℃ for 5min, on ice for 5min, added with 1ml of LB, and left at 30 ℃ for 3 h. And (3) keeping 100 mu L of supernatant, uniformly mixing and precipitating at 15000rpm for 30s, coating on a solid LB culture medium containing antibiotics, and standing at 30 ℃ for 4d to grow a single colony.

Selecting a single colony, shaking the single colony in an LB culture medium overnight, centrifuging the colony, removing a supernatant, collecting the colony, suspending the colony by using MMA, injecting Benzenbach for transgenically expressing mGFP5, injecting agrobacterium transformed by pKSE401-Cas9 vector capable of expressing Cas9 in an injection area on the 3 rd day after injection, and taking injection leaf Western blotting on the 6 th day after injection to detect the accumulation of SMV CP (figure 4).

Extracting DNA from injection leaves, carrying out PCR amplification on mGFP5 by using primers mGFP5/F and mGFP5/R, then carrying out enzyme digestion on a product by using restriction enzyme NdeI overnight, carrying out phenol-copy extraction and ethanol precipitation on the enzyme-digested product, and then carrying out agarose gel electrophoresis to detect the gene editing efficiency (figure 5) to find that the gene editing efficiency of version 7 (SMV-HHrz-gRNA-insertion sequence #3) containing the hammerhead ribozyme and the insertion sequence #3 is highest and that of version 8 (SMV-HHrz-gRNA-insertion sequence #4) containing the hammerhead ribozyme and the insertion sequence #4 is next to the gene editing efficiency of the version 7 (SMV-HHRz-gRNA-insertion sequence # 3); and (3) connecting the product after enzyme digestion with a T vector for sequencing after tapping and purifying, and comparing the sequencing result with the original gene sequence (figure 6 and figure 7) to verify that the mGFP5 of the injection leaf is really edited by the gene.

Agrobacterium transformed with a Cas 9-expressing pKSE401-Cas9 vector was injected into the upper leaf at day 9 after injection, and then the upper leaf injected with Cas9 was taken 3 days later (day 12 after injection) to carry out Western blotting to detect SMV CP accumulation (fig. 8), and all versions 1 to 8 were found to move systematically, with the highest accumulation of virus in version 7 (SMV-HHRz-gRNA-insert #3) and the next to version 8 (SMV-HHRz-gRNA-insert #4) containing hammerhead ribozyme and insert # 3.

Extracting DNA of the upper leaves of clone versions 7 and 8, carrying out PCR amplification on mGFP5, then carrying out enzyme digestion on the product overnight by using restriction enzyme NdeI, carrying out phenol-copy extraction and ethanol precipitation on the enzyme-digested product, and detecting the gene editing efficiency by agarose gel electrophoresis (figure 9); the product after enzyme digestion is subjected to tapping purification and then is connected with a T vector for sequencing, the sequencing result is subjected to sequence comparison with the original gene sequence (figure 10, figure 11), and the systematic leaf mGFP5 of the versions 7 and 8 is verified to be actually edited by the gene.

The invention simultaneously carries out an experimental process similar to that of the embodiment 1 aiming at the soybeans, obtains similar results and successfully realizes the gene editing of the soybeans.

The primers used in this example are shown in the following table:

TABLE 1 primer sequences used in example 1

Example 2

In this embodiment, a CRISPR/Cas9 gene editing system using a potyvirus as a vector is constructed, and as shown in fig. 12, the construction process is as follows:

1. selecting a potato Y virus ZT5 isolate (Genbank Access #: MF960848) as a raw material;

2. a nucleic acid sequence encoding a capsid protein of a codon-optimized potyvirus ZT5 isolate. Species selection common tobacco is optimized by one key by using an online website (https:// www.vectorbuilder.cn/tool/codon-optimization. html) to obtain an optimized nucleic acid sequence (SEQ ID NO.49), and the optimized potato Y virus capsid protein nucleic acid sequence is directly subjected to gene synthesis.

3. The potato Y virus is divided into 5 segments for PCR amplification, and meanwhile, a hammerhead ribozyme, a gRNA (editing PDS) and an insertion sequence are integrated into the infectious clone of the potato Y virus in a PCR mode, and the specific cloning steps are as follows:

(1) using potato virus Y infectious DNA clone as a template, amplifying a fragment 1 by using primers SMV/PVY-frag1-F and PVY-frag1-R, amplifying a fragment 2 by using primers PVY-frag2-F and PVY-frag2-R, amplifying an intermediate product by using primers PVY-frag3-F and PVY-frag3-R1, recovering the intermediate product by tapping rubber, using the recovered intermediate product as a template, and amplifying a fragment 3 by using primers PVY-frag3-F and PVY-frag 3-R2;

(2) using the optimized potato Y virus capsid protein nucleic acid sequence synthesized by the gene as a template, and amplifying a fragment 4 by primers PVY-frag4-F, PVY-frag4-R1, PVY-frag4-R2, PVY-frag4-R3, PVY-frag4-R4, PVY-frag4-R5 and PVY-frag 4-R6; using potato virus Y infectious DNA clone as a template, and amplifying a fragment 5 by primers PVY-frag5-F and SMV/PVY-frag 6-R;

(3) fragment 4 was mixed with fragment 5 as a template, and amplified by primers PVY-frag4-F and SMV/PVY-frag6-R to give fragment 4+ 5.

(4) The StuI and SmaI double-digested pCB301-314 and fragments 1,2,3, 4+5 are mixed, and the potato virus Y infectious DNA clone with inserted gRNA is obtained through yeast homologous recombination (the cloning construction scheme is shown as D in figure 3).

4. The procedures for extracting yeast plasmid and transforming agrobacterium C58C1 are the same as those for constructing CRISPR/Cas9 gene editing system (example 1) using soybean mosaic virus as vector.

5. After single colonies grow out, single colonies are picked and shaken in an LB culture medium overnight, after centrifugation, the supernatant is removed, and the thalli are collected, after the thalli are suspended by MMA, Benzenbach for expressing spCas9 transgenically is injected, and after injection, Western blotting is taken from injection leaves on the 6 th day to detect the accumulation of PVY CP (figure 13), and the PVY inserted with hammerhead ribozyme and gRNA can reach the replication level consistent with wild PVY.

Extracting DNA from an injection leaf, carrying out PCR amplification on a product of the injection leaf by using a primer PDS/F and a PDS/R, carrying out enzyme digestion on the product of the amplified PDS by using a restriction endonuclease NcoI overnight, carrying out phenol-copy extraction and ethanol precipitation on the enzyme-digested product, detecting the gene editing efficiency by agarose gel electrophoresis (figure 14), and showing that a part of PDS gene fragments amplified from plant tissues infected by PVY-HHRz-gRNA-complete CP can not be subjected to enzymolysis, thereby prompting that the part of PDS gene is edited; the product after enzyme digestion is subjected to tapping purification and then is connected with a T vector for sequencing, the sequencing result is subjected to sequence comparison with the original gene sequence (figure 15), and the fact that the injection leaf PDS is edited by the gene is verified;

western blotting of systemic leaves at day 12 after injection was performed to detect PVY CP accumulation (FIG. 16); extracting DNA of system leaves, carrying out PCR amplification on PDS, carrying out enzyme digestion on a product overnight by using restriction enzyme NcoI, carrying out phenol-copy extraction and ethanol precipitation on the enzyme-digested product, and detecting the gene editing efficiency by agarose gel electrophoresis (FIG. 17); the product after enzyme digestion is subjected to tapping purification and then is connected with a T vector for sequencing, the sequencing result is compared with the original gene sequence (figure 18), and the fact that the system leaf PDS is edited by the gene is verified. The invention also aims at the potatoes to carry out an experimental process similar to that of the embodiment 2, obtains similar results and successfully realizes the gene editing of the potatoes.

The sequences of the primers used in this example are shown in the following table:

TABLE 2 primer sequences used in example 2

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of Nanjing university

<120> gene editing vector, and method and application for editing gene thereof

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aaattaaaac aactcataaa gacaacaacg attacaaacg cattcaagac ttctatttct 60

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aaatcaactg aaatggcaac aatcatgttt ggagacttta ctgtgcagct gaagcataac 180

acaaagactg agaagagaaa gcgggtggtg gaaaccacca agcttgaaaa ggaggtgcgc 240

atggaaactg tccacgtgca agttatggag agtattactg taggttgctc agcacgctgt 300

gcgggcttga gcgcgtacac aaagtcgtcc cttaggaaag caatgaagga aggggatctg 360

agcgcgtcag ggggatgcca ttattgcggt cttcgggcat tggttggtga gggtcgcaaa 420

agggtgattt ctgtacccag gttggtggcg caacagaagg aagtggttgt cactaaggaa 480

gttcctcatt tctatgagga ggaatatgaa gttgaaatac catgtgtgac cactgagata 540

gcgcaaccag tagtggctgt cacttctatg agcaatgttt gcggaactgc tatgcagaca 600

aaggtgacaa gcactatcgt caccaaagat atgatggcga catctaagcc atcattgaag 660

caagtcagtc gtgctcttgt actgactggt aagaaagagg ttggtagcta tgacttggct 720

atcaagaaga tggatgaggc aatgcagcaa aattctgcgc tgcaaaaaca gctgttcatt 780

caacagcaaa gcaccattca acagaaacct aaaggagctg ttcaactgag gttatgctcg 840

tacgaacaag caaagaaacg tgttgaattg gcgcgtaaga gacaagaaga agaggaagat 900

tttctcaatg ggaagtatga acaacaattc tacgctggcg catccactcc aaagcctatg 960

aaatttgaag gagggagtgt tggttttaga acaaagtact ggagaccaac tccaaagaag 1020

attacagaaa ggcgtgcaac accacagtgt agaaaactaa catatgtctt ggaggaggtt 1080

ctctctttag cttccaagag tggcaagctg gttgaattta tcacaggagg caaaggaaag 1140

agtgtcaaag tttgttacat gcggaagcat ggcgcaatat tgcccaagtt ctctcttccg 1200

catgaagaag gtaggtatat ccatcaggag ctccagtatg aaagcatata cgagtttctt 1260

ccttatattt gcatgtttgc aaagtataag agcataagtg cggatgatat aacttatgga 1320

gatagtggtt tactgtttga tgagcgatca tctttaacca caaatcatac taaattaccg 1380

tactttgttg tccggggaag agagaatggg aaacttatta acgctcttga gatggttgtg 1440

agcatgaggg acatccagca ttattcccaa aatcctgagg ttcagttttt ccgtggttgg 1500

aaaaaggtgt tcgacacaat gcttccacat gtggagaatc atgaatgcac cattgatttc 1560

acaaatgagc agtgtggtga attggcagca gcaattaatc aatcaatctt tccagttaag 1620

aagctatcat gtaatcaatg tcgacagcac attaagaatc ttagttggga ggagtacaaa 1680

caattccttt tagcccacat gagttgtcat aggactgaat gggaagactt ccagaaagtt 1740

gatggcatga ggtatgtgaa gaaagtgatt gagacatcaa ctgcggaaaa cgcaagtctt 1800

cagacatcaa tggagattgt gcgtttaaca caaaactata agagtactca catgcttcaa 1860

atacaggata ttaataaggc tcttatgaag ggtccgtcgg ttacacaggg tgagctggag 1920

caagcttcca agcagctact agcaatgaca cagtggtgga aaaatcacat ggctttaact 1980

gatgaggatg cacttaaagt gttcaggaac aagagatctt ccaaagcact acttaaccca 2040

agtttgcttt gtgataatca gttggacaag aatggtaatt tcgtctgggg agagcgtggc 2100

aggcattcaa agcgattctt ttcgaattac tttgaagagg ttgttccttc tgaagggtac 2160

agtaagtatg tgattagaaa gaatccaaac ggacaaagag agttggcaat tgggtctctt 2220

attgtgccgc tagattttga gcgtgctcga atggcactgc agggcaaaag tgtaacaaga 2280

gagccaatta caatgtcatg tatctcaaga caagatggga actttgtgta tccttgctgt 2340

tgcgtcacac atgatgatgg aaaagctttc tattctgagc ttaagagtcc tacaaagcgc 2400

cacttggtta ttggaacatc tggtgatcca aaatacattg atctgccagc cactgatgca 2460

gataggatgt atataactaa agaaggattt tgctacctca acatcttctt ggcaatgttg 2520

gtcaatgtaa atgaagatga agccaaagac ttcacgaaga tggtaaggga tgttattgtg 2580

ccaaggctag gaaagtggcc gacaatgtta gatgtagcaa cagctacata catgctaaca 2640

gtttttcatc ctgaaaccag aaatgctgag cttccacgta ttttagttga ccatgcgtgt 2700

caaaccatgc atgtaataga ctcttttggg tccctcacag ttgggtacca tgttcttaaa 2760

gctggcacag tgaaccaatt gattcaattt gcttccaatg accttcagag cgagatgaaa 2820

ttttacagag ttggtggcga agtgcagcaa aggatgaagt gtgaaacagc acttataaca 2880

agtattttca aacctaagag gatgattcaa atccttgaga atgatccata cattcttttg 2940

atgggcctgg tttcaccttc tatcctgatt cacatgtatc gcatgaagca tttcgagaaa 3000

ggggtggagt tgtggataag taaggaacat agtgtggcaa agatcttcat tatattggaa 3060

cagctcacca agagggttgc tgcaaatgac gtgctacttg agcagcttga aatgatttca 3120

gagacttcag agagattcat gagcatatta gaggactgtc cccaagtacc accgtcatac 3180

aagacagcaa aagatttgtt aacaatgtat atagaaagaa aagcatccaa cagccagctt 3240

gttgagaatg gttttgtgga tatgaatgac aagttgtaca tggcatatga aaaaatctat 3300

tcagatcgct tgaagcagga atggcgcgca ttaagctggt tggaaaaatt ttctataaca 3360

tggcagttga aaagatttgc tccacatacg gagaaatgtt tgacaaagaa agttgtagaa 3420

gaaagcagcg catcttcagg aaactttgcg agtgtgtgct tcatgaatgc ccagtcacac 3480

ctcagaaatg taagaaatac actttttcaa aaatgtgacc aggtttggac tgcatcggtg 3540

cgagcttttg tgaggctcat aatttcgaca cttcataggt gctacagtga tattgtttat 3600

ctggtgaaca tctgtataat cttttcattg cttgtccaaa tgactagtgt actgcaaggc 3660

attgtcaaca cagcaaggag agataaagca ctcttaaata gatggaaaag gaaagaagat 3720

gaagaggccg tgatccattt gtatgaaatg tgtgaaaaga tggaaggtgg acacccaagg 3780

cttgagaaat ttttagacca tgtcaagggt gttagacctg atctactccc catagcagtg 3840

agcatgacag gacaatcaga agatgtctcc gcacaggcca aaacagcaac tcaattgcaa 3900

cttgagaaaa ttgtggcatt catggctttg ttgaccatgt gtattgataa tgaaaggagt 3960

gatgcggttt tcaaaatatt gagcaagtta aaggcatttt tcagcacaat gggtgaggat 4020

gttaaagtgc agagtcttga tgagattcaa aacattgatg aagacaagag gctcacaatt 4080

gatttcgacc ttgaaacaaa taaggagtct tccagtgttt cttttgatgt taaatttgag 4140

gcctggtgga atagacagtt ggaacagaat agagtaattc cacactacag gtcgacaggt 4200

gagtttcttg agttcacaag agaaacagca gccaaagttg caaatttgat agcaacatca 4260

agccacacag aatttttgat tcgaggtgca gttggctcag ggaaatcaac aggtttacca 4320

catcacctct caaagaaggg caaagttctg ctactagagc caactagacc gttagcggag 4380

aatgttagta agcagttgag ctttgaacct ttctatcata atgtaacact gagaatgaga 4440

ggattgagca agtttggctc aagtaacata gttgtcatga caagtgggtt tgcgtttcat 4500

tactatgtca acaatccaca acagctatct gatttcgatt ttatcataat agatgagtgc 4560

catgttcaag atagcccaac gattgcattc aactgtgcgc ttaaagaatt tgaatttagt 4620

ggcaaactta taaaagtgtc tgcaacacct ccaggaagag agtgcgaatt cacaacacaa 4680

catccagtga agttgaaagt tgaagaccat ttgtcttttc agaactttgt gcaagctcag 4740

ggtacaggat caaatgctga tatgatccaa catgggaaca atttactcgt atacgttgca 4800

agttacaatg aagttgacca attgtcacga ctattaactg agaaacatta caaggtgaca 4860

aaggttgatg ggagaacaat gcaaatggga aatgtagaga ttgcaaccac aggcacggaa 4920

gggaaaccac acttcatagt cgcaacaaat atcattgaga atggagtgac tcttgatatt 4980

gattgcgtaa ttgattttgg acttaaagtg gtggcaaccc ttgacacaga taaccggtgt 5040

gtgcgttaca acaaacagtc agtttcttat ggggagcgga tccagagact tggcagagtt 5100

ggtcgttgta aacctggatt tgcgctaagg attggacaca cgggaaaagg agttgaggaa 5160

gttcccgagt tcatagctac agaggcagcc tttctatcct ttgcttatgg gttgccagtt 5220

acaacacaaa gtgtctcgac caacatactg tcccgttgca cagtgaaaca agctcgagta 5280

gctctgaatt ttgagctaac tccatttttc accactaact tcataaagta tgatggtagc 5340

atgcacccag aaattcacag actgctcaag tcctacaaac taagggagtc cgagatgtta 5400

ttgaccaagt tagccatacc atatcagttt gttgggcagt gggtgacagt caaggagtat 5460

gaacgtcaag gtatccacct caattgtcca gagaaagtga aaataccttt ctatgtgcat 5520

ggaataccag ataagttgta cgagatgttg tgggacacag tttgtaaata caagaatgat 5580

gctggatttg gctcaattaa gagtgtgaat gcaacgaaga ttagttacac tctaagcact 5640

gacccgacag caattcctcg cacacttgca atactggacc atttgttgag tgaggagatg 5700

actaagaaga gtcattttga cacaattggc tctgctgtta ctgggtattc cttttctctt 5760

gcaggcatag ctgatggatt tagaaagagg tatttaaagg actacacaca gcataatata 5820

gccgtcctac aacaggctaa agcacagctg ctagagttcg actgcaacaa agttgacatc 5880

aacaacctgc acaatgttga gggtataggt attttaaatg cagtccaact acagagcaaa 5940

catgaggtga gcaaattttt gcagcttaaa ggaaagtggg atggaaagaa attcatgaat 6000

gatgctgttg tggctatctt cactttagtg ggcggtggtt ggatgctatg ggattacttc 6060

acaagagtca tacgtgaacc agtatcaact caaggaaaga agaggcagat acaaaagctc 6120

aaatttaggg atgcctttga cagaaaagta ggccgtgagg tgtatgcaga tgattacacc 6180

atggagcaca cctttgggga ggcatatacc aagaaaggaa agcagaaagg cagcactcgt 6240

acaaaaggaa tgggtcgcaa atcgaggaac ttcatacatc tatatggagt tgagccagag 6300

aattacagca tgattaggtt tgtagaccct ctaactgggc acacaatgga tgaacacccc 6360

agagttgata tcagaatggt tcaacaagag tttgaggata taaggaagga catgattggg 6420

gagggtgaat tggatcggca aagagtttac cataaccctg gtttacaggc ttatttcatt 6480

gggaagaata cagaggaagc actcaaagtt gacctcacac cacacagacc cacacttctc 6540

tgtcaaaaca gcaatgctat agcgggtttt ccagagaggg aggatgaatt gcgtcagaca 6600

ggcttgccac aagtagtttc caggtcagac gttccacgtg ccaaagaaag ggttgaagtg 6660

gaaagcaaat ctgtttacaa aggactcaga gattatagtg gcatttccac attaatatgt 6720

caacttacaa attcatcaga tggacacaaa gaaacaatgt ttggggttgg ctatggttct 6780

ttcattatca caaatgggca cttgtttagg aggaacaatg gaatgctcac agtcaagaca 6840

tggcatggtg agtttgtgat acacaacact acacagctca agatacattt tattcaaggg 6900

aaggatgtga ttctgattcg catgccaaag gactttcctc catttgggaa gcgcaatctc 6960

tttagacaac caaagcgtga ggaacgggtt tgtatggttg gaacaaactt tcaagagaag 7020

agcttgcgtg caacagtttc agaatcttct atgatattgc cagaggggaa aggttctttt 7080

tggatacatt ggattacaac ccaagatggt ttttgtgggt tgcctcttgt ttctgttaat 7140

gatgggcaca ttgttggaat acatggatta gcatctaatg attcagagaa gaactttttc 7200

gttccactca ctgatggatt tgagaaggaa tatctggaga atgctgataa cttgtcatgg 7260

gataagcact ggttttggga accaagcaag atagcatggg gctctttgaa tttagttgag 7320

gaacaaccaa aagaggagtt caaaatatca aagcttgtat cggatctctt tggaaacaca 7380

gtgacagtac aagggagaaa ggaaagatgg gttttagatg caatggaagg caacttagtg 7440

gcttgtgggc aagctgacag tgcattggta acaaagcatg ttgttaaagg aaagtgcccc 7500

tatttcgcac aatacctttc agtgaatcaa gaagcaaagt ccttctttga accacttatg 7560

ggtgcgtatc aaccaagccg attaaacaaa gatgcattca aacgagattt cttcaaatac 7620

aacaaaccag ttgttttgaa tgaagttgat ttccagtctt ttgagagggc agtggctgga 7680

gtgaagctga tgatgatgga atttgatttc aaggagtgtg tgtatgtgac tgatcctgat 7740

gaaatatacg actccttgaa catgaaagct gcagttggtg cacaatacaa agggaagaag 7800

caagattact tctctggaat ggatagtttt gacaaggaac gcttgcttta cctcagttgc 7860

gaaaggttat tttatgggga aaaaggagtg tggaatggat ctctgaaagc agagctaagg 7920

ccaattgaaa aagtgcaagc aaacaaaact agaacattca cagcagcacc aattgacaca 7980

ctacttggag caaaagtttg tgttgatgat ttcaacaatc aattttacag tctcaatctt 8040

acatgtccat ggacagttgg aatgaccaaa ttttatagag gttgggacaa gttgatgaga 8100

agtttacccg atggatgggt gtactgtcat gcagatggct cacagtttga tagttccctg 8160

acacctttac tactgaatgc agttcttgat gtcaggagct ttttcatgga agactggtgg 8220

gttggaagag aaatgctaga aaacctctat gctgagatag tctacacacc aatcttagca 8280

cctgatggca caatttttaa aaagttcaga ggaaacaaca gtgggcaacc atctacagtt 8340

gtggataaca ctttgatggt agtcattgcc atgtactatt cttgttgtaa gcatgggtgg 8400

tcagaggagg atattcagga aagattagtg tttttcgcca atggcgatga catcattctt 8460

gcagttagtg ataaggatac atggctatat gacactctta gcacttcgtt cgctgaactt 8520

ggtctcaatt acaactttga ggaacggaca aagaagaggg aggaattgtg gttcatgtct 8580

cacaaagcca tgttagttga tggaatttat attccaaaac ttgaacctga gagaattgtc 8640

tctattctag agtgggacag gagcaaagag cttatgcatc gcactgaggc aatatgtgca 8700

tcaatgattg aggcatgggg atacactgaa ttactgcaag agatccgcaa attttatttg 8760

tggcttttga acaaggatga gtttaaggag ctcgcttcgt ctggaaaagc accatatatt 8820

gcagagacag ctttgagaaa gctgtacaca gatgtcaatg cacaaacaag tgagctacaa 8880

agatatcttg aagtgttgga ctttaatcat attgatgact gttgtgaatc agtgtctcta 8940

caatctggta aagaaaaaga gggtgatatg gacgctgaca aggaccctaa aaaatctaca 9000

tcttcttcaa aaggtgccgg gacctctagt aaggacgtta acgtgggttc taagggtaaa 9060

gttgtcccaa gactccaaaa aataaccaga aaaatgaact tacctatggt cgaaggaaaa 9120

attatactgt cattagatca tttactcgaa tacaagccaa accaagtgga cctctttaac 9180

acaagggcta ctaggactca atttgaggca tggtataacg ctgtgaagga cgagtacgaa 9240

ttagacgacg agcaaatggg agttgtgatg aacggattta tggtctggtg tatagataac 9300

ggaacctcac ctgacgctaa cggagtttgg gttatgatgg acggtgagga gcaaatcgag 9360

taccccttga agcctatcgt ggagaacgcc aagcccacat tacgacagat tatgcatcat 9420

tttagcgacg ccgctgaggc ctatatcgaa atgaggaaca gtgagtcccc ttacatgccc 9480

aggtacggct tgcttcgtaa cctcagggac agggaacttg ctcgatacgc attcgacttt 9540

tacgaagtca caagtaagac tcctaataga gcccgtgagg ctatcgcaca aatgaaagcc 9600

gctgcattgt ctggcgtaaa taataaatta ttcgggttgg acggaaatat ttctacaaat 9660

tcagagaaca cagagcgtca tacagctaga gacgtcaacc agaatatgca tacactcctc 9720

gggatgggtc ctcaacaata acaagcactg atgagtccgt gaggacgaaa agagtaagct 9780

cttctgcttg ggctgcggat acccagatca tatgaaggtt ttagagctag aaatagcaag 9840

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 9900

caggcaagga gaaggaagga gacatggatg cagataaaga tccaaagaag agcaccagta 9960

gtagcaaggg agctggaaca agtagcaaag atgtaaatgt tggatcaaaa ggaaaggtgg 10020

ttccgcgttt gcagaagatt acaaggaaga tgaatcttcc aatggttgaa gggaagatca 10080

ttctcagttt ggaccacttg cttgagtata aacctaatca ggttgattta ttcaataccc 10140

gggcaacgag aacacagttc gaagcgtggt acaatgcagt taaagatgaa tatgagcttg 10200

atgatgaaca gatgggtgtg gttatgaatg gtttcatggt atggtgcatt gacaatggca 10260

catctccaga tgcaaatggc gtgtgggtga tgatggatgg agaagaacag attgaatatc 10320

cgctgaaacc cattgttgaa aatgcaaaac caactttgag acaaatcatg caccacttct 10380

cagatgcagc agaagcttac attgagatga gaaattctga aagtccgtat atgcctagat 10440

atggactact gaggaatttg agagatagag agctagcccg ctatgctttt gatttctatg 10500

aggttacttc caaaacacca aacagggcaa gggaagcaat agcgcagatg aaggctgcag 10560

ctctttcggg agttaacaac aagttgtttg gacttgatgg gaacatctca actaactccg 10620

aaaatactga aaggcacact gcaagggatg tgaatcaaaa catgcacact cttttgggta 10680

tgggcccaca gcagtaaagg ctaagtaaat tggtcacagt tatcatttcg ggtcgcttta 10740

tagtttgcta taatatagta tttgcactgt ctttaagtat agtgtgattg catcaccaaa 10800

taatgctttt gtttagtgtg gttttaacca cccagtgtgc tttatgttat agtttatgaa 10860

tggcagggag aaccattgtg ttgctggagc cctttggaga gtggttttaa ccacgtctag 10920

tggccgaggt acggcaatgt ttgttgtcct 10950

<210> 4

<211> 10665

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaattaaaac aactcaatac aacataagaa aatcaacgca aaaacactca caaaagcttt 60

caactctaat tcaaacaatt tgttaagttt caatttcaat cttcatcaaa caaactcttt 120

caatttcagt gtaagctatc gtaattcagt aagttatttc aaactctcgt aaattgcaga 180

agatcatcca tggcaactta cacatcaaca atccagtttg gttccattga atgcaaactt 240

ccatactcac ccgccccttt tgggctagtt gcggggaaac gagaagtttc aaccaccact 300

gaccccttcg caagtttgga gatgcagctg agtgcgcgat tacgaaggca ggagtttgca 360

actattcgaa catccaagaa tggtacttgc atgtatcgat acaagactga tgtccagatt 420

gcgcgcattc aaaagaagcg cgaggaaaga gaaagagagg aatataattt ccaaatggct 480

gcgtcaagtg ttgtgtcgaa gatcactatt gctggtggag agccaccttc aaaacttgaa 540

tcacaagtgc ggaggggtgt catccacaca actccaagga tgcgcacagc aaaaacatat 600

cacacgccaa agttgacaga gggacaaatg aaccacctta tcaagcaggt gaagcaaatt 660

atgtcaacca aaggagggtc tgttcaactg attagcaaga aaagtaccca tgttcactat 720

aaagaagttt tgggatcaca tcgcgcagtt gtttgcactg cacatatgag aggtttacga 780

aagagagtgg actttcggtg tgataaatgg accgttgtgc gtctacagca tctcgccagg 840

acggacaagt ggactaacca agttcgtgct actgatctac gcaagggcga tagtggagtt 900

atattgagta atactaatct caaaggaaac tttgggagaa gttcggaggg cctatttata 960

gtgcgtgggt cgcatgaagg aaaaatctat gatgcacgtt ccaaggttac ccaaggggtt 1020

atggactcaa tgattcagtt ctctagcgct gaaagctttt ggaagggatt ggacggcaat 1080

tgggcacaaa tgagatatcc tacagatcat acatgtgtgg caggcttacc agttgaagac 1140

tgtggcagag ttgcagcgat aataacacac agtattttac cgtgctataa aataacctgc 1200

cctacctgcg cccaacaata tgccaacttg ccagccagtg acttacttaa gatattacac 1260

aagcacgcaa gtgatggtct aaatcgattg ggggcagaca aagatcgctt tgtgcatgtc 1320

aaaaagttct tgacaatctt agagcactta actgaaccgg ttgatctgag tctagaaatt 1380

ttcaatgaag tattcaagtc tataggggag aagcaacaat cacctttcaa aaacctgaat 1440

attctgaata atttcttttt gaaaggaaag gaaaatacag ctcgtgaatg gcaggtggct 1500

caattaagct tacttgaatt ggcaagattc caaaagaaca gaacggataa tatcaagaaa 1560

ggagacatct cgttctttag gaataaacta tccgccaaag caaattggaa cttgtatctg 1620

tcatgtgata accagctgga caagaatgca aatttcctgt ggggacagag ggaatatcat 1680

gctaagcgat ttttctcgaa ctatttcgag gaaattgatc cagcgaaggg ctattcagca 1740

tacgaaaatc gtttgcatcc gaatgggaca agaaaacttg caattggaaa cctaattgta 1800

ccacttgatc tggctgagtt taggcggaag atgaaaggtg attataaaag acagccaggg 1860

gtgagtaaga agtgcacgag ctcgaaggat ggaaactacg tgtatccctg ttgttgcact 1920

acacttgatg atggctcagc tgttgaatca acattttacc cgccaactaa gaagcacctc 1980

gtaataggta atagtggcga ccaaaagtat gttgacttac caaaagggaa ttctgagatg 2040

ttatatattg ccaggcaagg cttctgttac attaacattt tcctcgcgat gttgattaac 2100

attagtgagg aagatgcaaa ggatttcact aagaaggttc gtgacatgtg tgtgccaaag 2160

cttggaacct ggccaaccat gatggatctg gctacaactt gtgctcaaat gagaatattc 2220

taccctgatg ttcatgatgc agaactgcct agaatactag tcgatcacga aacgcagaca 2280

tgccatgtgg ttgactcgtt tggctcacaa acaactgggt atcatatttt gaaagcatct 2340

agcgtgtccc aacttatttt gtttgctaat gatgagttgg agtctgacat taagcactat 2400

agagttggtg gtattcctgg agcatgctct gagcttgggt ccgcaatatc accttttaga 2460

gaaggaggaa tcataatgtc tgagtcagca gcgctaaaac tgctcctaaa gggaattttt 2520

agacctaagg tgatgagaca gttgctgtta gatgagcctt acctgttgat tctatcaata 2580

ttatctcctg gcatactgat ggctatgtat aataatggga tttttgaact tgcggtaagg 2640

ttgtggatta atgagaaaca atccatagcc atgatagcat cgctactatc agctttagcc 2700

ctacgagtgt cggcggcaga aacactcgtc gcacagagga ttatcattga tgctgcagct 2760

acagacctcc ttgatgctac gtgtgatggg ttcaacctac atctaacgta ccccactgca 2820

ttgatggtgt tgcaagttgt taagaataga aatgaatgtg atgataccct attcaaggcg 2880

ggtttttcaa gttacaacac gagcgtcgta cagattatgg aaaaaaatta tctaaatctc 2940

ttggacgatg cttggaaaga tttaacttgg cgggaaaaat tatccgcaac atggtactca 3000

tacagagcaa aacgctctat cactcggtac ataaaaccca caggaagggc agatttgaga 3060

gggttataca acatatcacc acaagcattc ttgggccgaa gcgcccaggt ggtcaaaggt 3120

actgcctcag gattgagcga gcgatttaat aattatttca atactaagtg tgtaaatatt 3180

tcatcctttt tcattcgtag aatctttagg cgtttgccaa cttttgtcac ttttgttaac 3240

tcattattag ttattagtat gttgactagc gtagtggcag tgtgtcaggc aataatttta 3300

gatcagagga agtataggag agaaatcgag ttgatgcaga tagagaagaa tgaaattgtc 3360

tgcatggagc tatatgcaag tttacagcgc aaacttgaac gcgatttcac atgggatgag 3420

tacattgagt atttgaaatc agtaaaccct cagatagttc agtttgctca agcgcagatg 3480

gaagaatatg atgtgagaca ccagcgttcc acaccaggtg ttaaaaattt ggaacaagtg 3540

gtagcattta tggctttagt catcatggtg ttcgatgctg aaaggagtga ttgcgtgttc 3600

aagactctca ataaatttaa gggtgtcctt tcctcaatgg accatgaagt tagacatcag 3660

tccttagacg atgtgatcaa gaactttgat gagaggaatg agattattga ttttgaattg 3720

agtgaggaca caattcgaac atcatcagtg ctagatacaa agtttagtga ttggtgggac 3780

cgacaaatcc agatgggaca tacacttcca cattacagaa ccgaggggca cttcatggaa 3840

tttacaagag caactgctgt tcaagtggct aatgacattg cccatagcga acacctagac 3900

tttctagtaa ggggagctgt tgggtctgga aagtcaactg ggttgcctgt tcatcttagt 3960

gtagctggat ctgtgctttt gattgaacca acgcgaccac tagcggagaa cgttttcaaa 4020

cagctatcta gtgaaccatt cttcaagaag ccaacactgc gtatgcgtgg aaatagcata 4080

tttggctctt ctccaatctc cgtcatgact agtggatttg cactacacta ctttgccaat 4140

aatcgctctc aattagctca gttcaacttt gtaatatttg atgagtgcca tgttctggat 4200

ccttccgcaa tggcgttccg cagtctgctg agtgtttatc atcaagcatg caaagtatta 4260

aaagtgtcag ctactccagt gggaagagag gttgaattta caacacagca accagtcaag 4320

ttaatagtgg aggacacact gtctttccaa tcatttgttg atgcacaagg ttctaaaact 4380

aatgctgatg ttgttcagtt tggttcgaac gtacttgtgt acgtgtcgag ctacaatgaa 4440

gttgatacct tggctaagct cctaacagac aagaatatga tggtcacaaa ggttgatggc 4500

agaacaatga agcacggttg cctagaaatt gtcacaaaag gaaccagtgc gagaccacat 4560

tttgttgtag caaccaacat aattgagaat ggagtgactt tggacataga cgtggttgta 4620

gattttgggt tgaaagtctc accgttcttg gacattgaca ataggagcat tgcttacaat 4680

aaggtgagtg ttagctatgg tgagagaatt caaaggctgg gtcgtgttgg acgcttcaag 4740

aaaggagtag cattgcgcat tggacacact gagaagggaa ttattgaaat tccaagcatg 4800

atcgctacag aggcggctct tgcttgcttt gcatataact tgccagtgat gacaggaggc 4860

gtctcaacta gtctgattgg caattgtact gtgcgccaag ttaaaacaat gcagcaattt 4920

gaattgagtc ccttctttat ccagaatttc gtcgcccatg atggatcaat gcatcctgtc 4980

atacatgaca ttcttaaaaa gtataaactt cgagattgta tgacaccttt gtgcgatcag 5040

tctataccat acagggcatc gagcacttgg ttatcggtta gtgaatatga gcgacttgga 5100

gtggccttag aaattccaaa gcaagtcaaa attgcattcc atatcaaaga gatccctcct 5160

aagctccacg aaatgctttg ggaaacggtt gtcaagtaca aagacgtttg cttatttcca 5220

agcattcgag catcgtccat cagcaaaatc gcatacacat tgcgtacaga cctcttcgcc 5280

atcccaagaa ctctaatatt ggtggagaga ctgcttgaag aggagcgagt gaagcagagc 5340

caattcagaa gtctcatcga tgaaggatgc tcaagcatgt tttcaattgt caatttgaca 5400

aacactctca gagctagata tgcaaaagat tacaccgcag agaacataca aaaacttgag 5460

aaagtgagaa gtcaattgaa agaattctca aatttggatg gttctgcatg tgaggaaaat 5520

ttaataaaga ggtatgagtc tttgcagttc gttcatcacc aggctacgac gtcacttgca 5580

aaggatctca agttgaaggg gacttggaag aagtcattag tggccaaaga cttgatcata 5640

gcaggcgctg ttgcaattgg tggaatagga ctcatatata gttggttcac acaatcagtt 5700

gagactgtgt ctcaccaagg gaaaaataaa tccaaaagaa ttcaagcctt gaagtttcgc 5760

catgctcgtg acaaaagggc tggttttgaa attgacaaca atgatgacac aatagaggaa 5820

ttctttggat ctgcatacag gaaaaaggga aaaggtaaag gtaccactgt tggtatgggc 5880

aagtcaagca ggaggtttgt taatatgtat ggatttgacc caacagaata ttcattcatc 5940

cagttcgttg atccgctcac tggagctcaa attgaagaga acgtctatgc tgatattaga 6000

gacatccaag agcgctttag tgatgtccgc aagaaaatgg tagaggatga tgaaatcgaa 6060

ttgcaagcat tgggcagcaa cacaaccatt catgcttact tcaggaaaga ttggtctgac 6120

aaggctctta aaattgattt gatgccacac aacccactca aaatctgtga taaatcgaat 6180

ggtattgcta agtttcctga aagagaactt gagttgaggc aaactgggcc agcaatagag 6240

gttgatgtga aagacattcc aaaacaggaa gtggagcatg aagccaaatc actcatgaga 6300

ggtttaaggg atttcaatcc aattgctcaa acagtttgca gagtaaaagt gtctgttgaa 6360

tatggaacgt ctgaaatgta tgggttcggt tttggtgcgt atattatagt aaaccaccat 6420

ctattcaaga gcttcaatgg atccatggaa gtgcgatcaa tgcatggaac attcagagtg 6480

aagaatttgc atagcttgag cgttttaccg atcaaaggca gagacattat catcataaag 6540

atgccaaagg atttccctgt tttcccacaa aaactgcact tccgagctcc agtgcagaat 6600

gagaggattt gtttggttgg aactaatttt caagaaaaac atgcatcatc aatcatcaca 6660

gaaacgagta ctacatacaa tgtaccgggc agcacttttt ggaagcattg gattgaaaca 6720

aatgatgggc attgtggatt accagtagtg agtacagctg atggatgtct agttggaata 6780

cacagcttgg cgaataatgt gcaaaccacg aattattatt cagcctttga tgaggatttt 6840

gaaagtaagt atctccgaac taatgagcat aatgagtgga ccaaatcgtg ggtatataac 6900

ccagatactg tgttgtgggg tccgttgaag ctcaaggaga gtacccctaa aggcctgttt 6960

aagacaacaa aacttgtaca ggatttaatt gatcatgatg ttgttgtaga gcaagctaaa 7020

cattctgcgt ggatgtatga ggctctaaca gggaatttgc aagctgtggc gacaatgaag 7080

agtcagctag tgacaaagca cgtggtcaaa ggggagtgtc ggcacttcaa agagttctta 7140

actgtggatt cggaagcaga agctttcttc aggcctttga tggatgctta tgggaagagc 7200

ttattaaata gagaagcata tataaaggac ataatgaaat actcaaagcc tattgatgtt 7260

ggaatagtag actgtgatgc ttttgaagag gctatcaata gggttatcat ttatctgcaa 7320

gtgcatggct tccagaaatg caattacatc accgatgagc aggaaatttt caaagctctc 7380

aatatgaaag ctgctgtcgg agctatgtat ggaggcaaga agaaagacta cttcgagcat 7440

tttactgagg cggataaaga ggaaattgtt atgcaaagtt gctttcgatt gtacaagggc 7500

tcgcttggca tatggaatgg atcattgaaa gcagaacttc ggtgcaaaga gaagatactt 7560

gcaaataaga caaggacatt cactgctgca cctttagata ctttattggg tggaaaggtg 7620

tgcgttgatg attttaataa tcaattctac tcaaagaaca ttgaatgctg ctggactgtt 7680

ggaatgacta agttttatgg aggttgggac aaattgcttc ggcgtctacc tgaaaattgg 7740

gtgtactgcg atgccgatgg ttcacaattc gatagttcac tcaccccata cctaattaat 7800

gctgttctca tcatcagaag cacatacatg gaagattggg acctggggtt gcaaatgttg 7860

cgcaatttgt acacagaaat aatttacaca ccaatctcaa ctccagatgg aacaattgtc 7920

aagaagttta gaggtaataa tagcggtcaa ccttctaccg ttgtggataa ttctctcatg 7980

gttgtccttg ctatgcatta cgctctcatt aaggagtgcg tcgagtttga agaaatcgac 8040

agcacgtgtg tattctttgt taatggtgat gacttattga ttgctgtgaa tccggagaaa 8100

gagagcattc tcgatagaat gtcacaacat ttctcagatc ttggtttgaa ctatgatttt 8160

tcgtcgagaa caagaaggaa ggaggaattg tggttcatgt cccatagagg cctgctaatc 8220

gagggtatgt acgtgccaaa gcttgaagaa gagagaattg tatccattct gcaatgggat 8280

agagctgatc tgccagagca cagattagaa gcgatctgtg cagcaatgat agaatcctgg 8340

ggttattttg agttaacgca ccaaatcagg agattctact catggttgtt gcaacagcaa 8400

cctttttcaa cgatagcaca ggaaggaaaa gctccataca tagcgagcat ggcattgaag 8460

aagctgtaca tggataggac agtagatgag gaggaactga aggctttcac tgaaatgatg 8520

gttgccttgg atgatgaatt tgagtgcgat acttatgaag tacaccatca agctaacgat 8580

actattgacg ctggtggttc ttctaaaaag gacgctaagc ctgaacagga ttctattcaa 8640

tctaatccaa ataagggtaa ggacaaagac gttaacgcag gaacttcagg aactcacaca 8700

gttccaagga ttaaagcaat tacttctaag atgaggatgc caaagtctaa aggtgctact 8760

gttcttaatc ttgagcatct tttggaatac gcacctcagc agatcgacat ctctaacaca 8820

agagctacac agtctcaatt cgacacttgg tacgaagctg ttagaatggc ttatgatatt 8880

ggtgagacag aaatgcctac agttatgaac ggattgatgg tgtggtgtat cgagaacggt 8940

acttctccta acgttaatgg tgtgtgggtg atgatggacg gagacgagca ggttgaatat 9000

ccacttaagc ctattgtgga aaacgctaag cctactttga gacagattat ggctcacttt 9060

tctgacgtgg ctgaggctta cattgagatg agaaataaga aagagcctta catgcctaga 9120

tacggactta tcagaaacct tagagacgtt ggacttgcta gatacgcttt cgatttctac 9180

gaagttactt ctagaactcc tgttagagca agagaggctc atatccagat gaaagctgct 9240

gctcttaagt ctgctcagcc aagattgttt ggacttgatg gaggaatttc tactcaggaa 9300

gaaaatactg aaagacatac tactgaagac gtttcacctt ctatgcacac acttttgggt 9360

gttaaaaata tgtaacaagc actgatgagt ccgtgaggac gaaaagagta agctcttctg 9420

cttgggctgc gtttggtagt agcgactcca tgttttagag ctagaaatag caagttaaaa 9480

taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgcaaatg 9540

acacaatcga tgcaggagga agcagcaaga aagatgcaaa accagagcaa gacagcatcc 9600

agtcaaaccc gaacaaagga aaagataagg atgtgaatgc tggtacatct gggacacata 9660

ctgtgccgag aatcaaggct atcacgtcca aaatgagaat gcccaaaagc aagggagcaa 9720

ccgtgctaaa cttagaacac ttgcttgagt atgctccaca acaaattgat atttcaaata 9780

ctcgggcaac tcaatcacag tttgatacgt ggtatgaggc agtgcggatg gcatacgaca 9840

taggagaaac tgagatgcca actgtgatga atgggcttat ggtttggtgc attgaaaatg 9900

gaacctcgcc aaatgtcaac ggagtttggg ttatgatgga tggggatgaa caagtcgagt 9960

acccgttgaa accaatcgtt gagaatgcga aaccaaccct taggcaaatc atggcacatt 10020

tctcagatgt tgcagaagcg tatatagaaa tgcgcaacaa aaaggaacca tatatgccac 10080

gatatggttt aattcgaaat ctgcgggatg tgggtttagc gcgttatgcc tttgactttt 10140

atgaggtcac atcacgaaca ccagtgaggg ctagggaagc gcacattcaa atgaaggccg 10200

cagcattgaa atcagcccaa cctcgacttt tcgggttgga cggtggcatc agtacacaag 10260

aggagaacac agagaggcac accaccgagg atgtctctcc aagtatgcat actctacttg 10320

gagtcaagaa catgtgatat agtgtctctc cggacgatat ataagtattt acatatgcag 10380

taagtatttt ggcttttcct gtactacttt tatcataatt aataatcagt ttgaatatta 10440

ctaatagata gaggtggcag ggtgatttcg tcattgtggt gactctatct tttaattccg 10500

cattattaag tcttagataa aagtgccggg ttgtcgttgt tgtggatgat tcatcgatta 10560

ggtgatgttg cgattctgtc gtagcagtga ctacgtctgg atctatctgc ttgggtggtg 10620

ttgtgattcc gtcataacag tgactgcaaa cttcaatcag gagac 10665

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacgtaaggg atgacgcaca atcc 24

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatgtctcaa tcactttctt cacatac 27

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

catgtggaga atcatgaatg cacca 25

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctttaacatc ctcacccatt gtg 23

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cagcaactca attgcaactt gagaaaattg 30

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtatctgcct cttctttcct tgagttgata c 31

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gttgacatca acaacctgca caatgttg 28

<210> 12

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccttgtcagc gtccatatca ccctcttttt ctttaccaga ttgtagagac actgattcac 60

<210> 13

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggtcccggca ccttttgaag aagatgtaga ttttttaggg tccttgtcag cgtccatatc 60

<210> 14

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttgatgactg ttgtgaatca gtgtctctac aatctggtaa agaaaaagag ggtgatatgg 60

<210> 15

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tatttctagc tctaaaacct tcatatgatc tgggtatctt attgttgagg acccatcccg 60

<210> 16

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agttgataac ggactagcct tattttaact tgctatttct agctctaaaa ccttcatatg 60

<210> 17

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac 60

<210> 18

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcttactctt ttcgtcctca cggactcatc agtgcttgtt attgttgagg acccatcccg 60

<210> 19

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cttcatatga tctgggtatc cgcagcccaa gcagaagagc ttactctttt cgtcctcacg 60

<210> 20

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tagccttatt ttaacttgct atttctagct ctaaaacctt catatgatct gggtatccgc 60

<210> 21

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caccgagtcg gtgctttttt gaggttactt ccaaaacacc aaac 44

<210> 22

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

caccgagtcg gtgctttttt attgagatga gaaattctga aagtccg 47

<210> 23

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

caccgagtcg gtgctttttt ttggaccact tgcttgagta taaacc 46

<210> 24

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

caccgagtcg gtgctttttt tcaggcaagg agaaggaagg ag 42

<210> 25

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

accggcaaca ggattcaatc ttaagaaac 29

<210> 26

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

taaaattatt gcctgacaca ctgccac 27

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gaatctttag gcgtttgcca ac 22

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

acagggaaat cctttggcat c 21

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gttttaccga tcaaaggcag agac 24

<210> 30

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tagaagaacc accagcgtca atagtatcgt tagcttgatg gtgtacttca taagtatcgc 60

<210> 31

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

attgaataga atcctgttca ggcttagcgt cctttttaga agaaccacca gcgtcaatag 60

<210> 32

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tgaatttgag tgcgatactt atgaagtaca ccatcaagct aacgatacta ttgacgctgg 60

<210> 33

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttttcgtcct cacggactca tcagtgcttg ttacatattt ttaacaccca aaagtgtgtg 60

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gcttactctt ttcgtcctca cggactcatc 30

<210> 35

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

atggagtcgc tactaccaaa cgcagcccaa gcagaagagc ttactctttt cgtcctcacg 60

<210> 36

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

actagcctta ttttaacttg ctatttctag ctctaaaaca tggagtcgct actaccaaac 60

<210> 37

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gttgataacg gactagcctt attttaactt gctatttcta g 41

<210> 38

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac 60

<210> 39

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

caccgagtcg gtgctttttt gcaaatgaca caatcgatgc ag 42

<210> 40

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atgagtaaag gagaagaact tttcactgga g 31

<210> 41

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ttatttgtat agttcatcca tgccatgtgt aatccc 36

<210> 42

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttaggttcac aagtgggaca atcttc 26

<210> 43

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

cagcatcaca ctttcgcatt caaaac 26

<210> 44

<211> 198

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

gaggttactt ccaaaacacc aaacagggca agggaagcaa tagcgcagat gaaggctgca 60

gctctttcgg gagttaacaa caagttgttt ggacttgatg ggaacatctc aactaactcc 120

gaaaatactg aaaggcacac tgcaagggat gtgaatcaaa acatgcacac tcttttgggt 180

atgggcccac agcagtaa 198

<210> 45

<211> 297

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

attgagatga gaaattctga aagtccgtat atgcctagat atggactact gaggaatttg 60

agagatagag agctagcccg ctatgctttt gatttctatg aggttacttc caaaacacca 120

aacagggcaa gggaagcaat agcgcagatg aaggctgcag ctctttcggg agttaacaac 180

aagttgtttg gacttgatgg gaacatctca actaactccg aaaatactga aaggcacact 240

gcaagggatg tgaatcaaaa catgcacact cttttgggta tgggcccaca gcagtaa 297

<210> 46

<211> 609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ttggaccact tgcttgagta taaacctaat caggttgatt tattcaatac ccgggcaacg 60

agaacacagt tcgaagcgtg gtacaatgca gttaaagatg aatatgagct tgatgatgaa 120

cagatgggtg tggttatgaa tggtttcatg gtatggtgca ttgacaatgg cacatctcca 180

gatgcaaatg gcgtgtgggt gatgatggat ggagaagaac agattgaata tccgctgaaa 240

cccattgttg aaaatgcaaa accaactttg agacaaatca tgcaccactt ctcagatgca 300

gcagaagctt acattgagat gagaaattct gaaagtccgt atatgcctag atatggacta 360

ctgaggaatt tgagagatag agagctagcc cgctatgctt ttgatttcta tgaggttact 420

tccaaaacac caaacagggc aagggaagca atagcgcaga tgaaggctgc agctctttcg 480

ggagttaaca acaagttgtt tggacttgat gggaacatct caactaactc cgaaaatact 540

gaaaggcaca ctgcaaggga tgtgaatcaa aacatgcaca ctcttttggg tatgggccca 600

cagcagtaa 609

<210> 47

<211> 798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tcaggcaagg agaaggaagg agacatggat gcagataaag atccaaagaa gagcaccagt 60

agtagcaagg gagctggaac aagtagcaaa gatgtaaatg ttggatcaaa aggaaaggtg 120

gttccgcgtt tgcagaagat tacaaggaag atgaatcttc caatggttga agggaagatc 180

attctcagtt tggaccactt gcttgagtat aaacctaatc aggttgattt attcaatacc 240

cgggcaacga gaacacagtt cgaagcgtgg tacaatgcag ttaaagatga atatgagctt 300

gatgatgaac agatgggtgt ggttatgaat ggtttcatgg tatggtgcat tgacaatggc 360

acatctccag atgcaaatgg cgtgtgggtg atgatggatg gagaagaaca gattgaatat 420

ccgctgaaac ccattgttga aaatgcaaaa ccaactttga gacaaatcat gcaccacttc 480

tcagatgcag cagaagctta cattgagatg agaaattctg aaagtccgta tatgcctaga 540

tatggactac tgaggaattt gagagataga gagctagccc gctatgcttt tgatttctat 600

gaggttactt ccaaaacacc aaacagggca agggaagcaa tagcgcagat gaaggctgca 660

gctctttcgg gagttaacaa caagttgttt ggacttgatg ggaacatctc aactaactcc 720

gaaaatactg aaaggcacac tgcaagggat gtgaatcaaa acatgcacac tcttttgggt 780

atgggcccac agcagtaa 798

<210> 48

<211> 82

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt tt 82

<210> 49

<211> 804

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

gctaacgata ctattgacgc tggtggttct tctaaaaagg acgctaagcc tgaacaggat 60

tctattcaat ctaatccaaa taagggtaag gacaaagacg ttaacgcagg aacttcagga 120

actcacacag ttccaaggat taaagcaatt acttctaaga tgaggatgcc aaagtctaaa 180

ggtgctactg ttcttaatct tgagcatctt ttggaatacg cacctcagca gatcgacatc 240

tctaacacaa gagctacaca gtctcaattc gacacttggt acgaagctgt tagaatggct 300

tatgatattg gtgagacaga aatgcctaca gttatgaacg gattgatggt gtggtgtatc 360

gagaacggta cttctcctaa cgttaatggt gtgtgggtga tgatggacgg agacgagcag 420

gttgaatatc cacttaagcc tattgtggaa aacgctaagc ctactttgag acagattatg 480

gctcactttt ctgacgtggc tgaggcttac attgagatga gaaataagaa agagccttac 540

atgcctagat acggacttat cagaaacctt agagacgttg gacttgctag atacgctttc 600

gatttctacg aagttacttc tagaactcct gttagagcaa gagaggctca tatccagatg 660

aaagctgctg ctcttaagtc tgctcagcca agattgtttg gacttgatgg aggaatttct 720

actcaggaag aaaatactga aagacatact actgaagacg tttcaccttc tatgcacaca 780

cttttgggtg ttaaaaatat gtaa 804