CN114853858A - Insect-resistant cyiron toxin protein gene, expression vector and application - Google Patents

Insect-resistant cyiron toxin protein gene, expression vector and application Download PDF

Info

Publication number
CN114853858A
CN114853858A CN202210324163.XA CN202210324163A CN114853858A CN 114853858 A CN114853858 A CN 114853858A CN 202210324163 A CN202210324163 A CN 202210324163A CN 114853858 A CN114853858 A CN 114853858A
Authority
CN
China
Prior art keywords
leu
ala
val
gly
asp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210324163.XA
Other languages
Chinese (zh)
Other versions
CN114853858B (en
Inventor
邹长松
王朋宝
张寿洲
黄锦岭
马建超
刘欢
常贯晓
李林洲
王思博
董珊珊
程祥飞
王磊
孙晓
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Xianhu Botanical Garden Shenzhen Garden Research Center
BGI Shenzhen Co Ltd
Henan University
Original Assignee
Shenzhen Xianhu Botanical Garden Shenzhen Garden Research Center
BGI Shenzhen Co Ltd
Henan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Xianhu Botanical Garden Shenzhen Garden Research Center, BGI Shenzhen Co Ltd, Henan University filed Critical Shenzhen Xianhu Botanical Garden Shenzhen Garden Research Center
Priority to CN202210324163.XA priority Critical patent/CN114853858B/en
Publication of CN114853858A publication Critical patent/CN114853858A/en
Application granted granted Critical
Publication of CN114853858B publication Critical patent/CN114853858B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Pest Control & Pesticides (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Insects & Arthropods (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention belongs to the technical field of genetic engineering, and discloses an insect-resistant cycadoxin gene, an expression vector and application thereof, wherein the nucleotide sequences of the insect-resistant cycadoxin gene and homologous genes thereof are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. The toxic protein gene provided by the invention is derived from cycados cycas, and has a toxic effect on diamondback moth and cotton bollworm larvae through exogenous expression injection. The gene is introduced into cotton, so that the cotton obtains the resistance of spodoptera frugiperda and cotton bollworm, and progeny can be stably inherited. The invention provides a new insect-resistant gene for the development of insect-resistant plants, which not only can lead receptors such as cotton and the like to obtain insect resistance of various mechanisms, but also can strengthen the insect resistance of transgenic cotton by utilizing a gene complementation mode, delay the generation of resistance of pests, widen the insect resistance spectrum of cotton and lead the cotton to obtain broad-spectrum insect resistance capable of efficiently killing main pests.

Description

Insect-resistant cyhalothrin gene, expression vector and application
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an insect-resistant cyhalothrin gene, an expression vector and application.
Background
Sustainable development of crops such as wheat, corn, cotton and the like is important for guaranteeing food safety in China. Among them, cotton is not only the most important natural fiber crop in the world, but also an important oil crop and high protein food crop.
As much as 40% of crop production worldwide per year is lost to pests, resulting in losses of at least $ 700 billion. In order to resist pest attack, scientists have made a series of important advances in the aspects of insect-resistant gene mining and resistance mechanism analysis. At present, insect-resistant genes which are successfully researched mainly comprise insect neurotoxins such as bacillus thuringiensis insecticidal genes, cowpea trypsin inhibitors, arrowhead protease inhibitors, amylase inhibitors, nutritional insecticidal proteins, cholesterol oxidase, galangal agglutinin, pinellia ternate agglutinin, amaranthus caudatus agglutinin, scorpion venom genes and the like. The most successful application on cotton is a new bivalent transgenic insect-resistant cotton variety developed by Guo Sanzhu and the like, and the genetically transformed insect-resistant genes of the cotton are a Bt insecticidal gene GFMCry1A and a cowpea trypsin inhibitor gene Cpti.
Although the existing transgenic cotton with insect-resistant genes can effectively control lepidoptera, coleopteran and homopteran pests, the genes are expressed all the time in the whole growth period of the cotton, so that the pests are likely to generate resistance to the genes, and the genes become the greatest threat in the development of transgenic cotton. Among them, Australian transgenic insect-resistant cotton has been ineffective against Helicoverpa armigera.
At present, available resistance gene resources for crop disease and insect resistant breeding are deficient, new disease and insect resistant genes are urgently needed to be excavated, the insect resistant capability of transgenic crops or plants is enhanced by utilizing a gene complementation mode, and meanwhile, the generation of resistance (tolerance) of pests is delayed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an insect-resistant cyhalothrin gene, an expression vector and application.
The invention is realized in such a way that the nucleotide sequence of the insect-resistant cyiron toxin protein gene is SEQ ID NO. 1.
The other purpose of the invention is to provide a homologous gene of the insect-resistant cycadoxin gene in cycads, wherein the nucleotide sequence of the homologous gene is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The invention also aims to provide a cycadoxin optimized gene obtained by codon optimization of the insect-resistant cycadoxin gene, wherein the optimized species is cotton, and the nucleotide sequence of the cycadoxin optimized gene is SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.
Another purpose of the invention is to provide a protein for coding the insect-resistant thifenuron protein gene, wherein the amino acid sequence of the protein is SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO. 15.
It is another object of the present invention to provide a pesticidal use or pesticide manufacture encoding said insect-resistant cyhalothrin gene, which range of applications include but are not limited to plant or crop pests such as cotton bollworm, diamond back moth, spodoptera frugiperda, nematodes and the like.
The invention also aims to provide a plant or crop expression vector constructed by applying the insect-resistant cyhalothrin gene, and a construction method of the expression vector comprises the following steps:
preparation of a linearized vector: the circular vector was linearized by double digestion of pVM062-3 FLAG vector with the restriction enzymes BstBI and MluI.
Amplification of target sequences with homology arms:
with the forward primer:
5’-gggcggtaccccgggttcgaaATGGTTATGGTAATTGACTATCCAAGTC-3’；
reverse primer: 5'-cactagttaattaagacgcgtCTATGTCATGCGTAGAATGGCCC-3'
Carrying out PCR amplification on the cyathrin gene as a primer.
And (3) detecting and recovering a vector enzyme digestion product and a PCR product: agarose gel electrophoresis is used for detecting the target gene fragment and the carrier enzyme digestion product
Figure BDA0003571196030000031
The Quick Gel Extraction Kit recovers the target fragment.
Glue recovery product recombination: use of
Figure BDA0003571196030000032
-Basic Seamless Cloning and Assembly Kit for homologous recombination in the following reaction system: 2 × Basic Assembly Mix 5 μ L; carrying out enzyme digestion on the vector X mu L; mu.L of PCR product; sterile water was added to 10 μ L. In a 10 μ L reaction system, the addition amount of the vector and each insert is 0.01-0.25 pmols, and the optimal molar ratio of the vector to each insert is 1: 2; the mixture was gently mixed and reacted at 50 ℃ for 15min to effect conversion. Sequencing and identifying: positive clones were selected by colony PCR and plasmid PCR for sequencing analysis.
Further, the amplification system is: KOD One TM PCR Master Mix 25. mu.L; upstream primer 10. mu.M each 1.5. mu.L; downstream primer 10. mu.M each 1.5. mu.L; 2 mu L of template; 20 mu L of sterilized water; the total volume was 50. mu.L.
The procedure for the PCR was: the PCR program is 98 ℃ for 5 min; 30 cycles of 98 ℃ for 30s, 50 ℃ for 30s and 68 ℃ for 3 min; storing at 68 deg.C for 10min and 4 deg.C.
Further, the transformation method comprises the following steps:
(1) thawing the Trans1-T1 competent cells on ice, standing for 2-5 minutes, observing whether the cells are thawed, and flicking the bottom of the tube 1-2 times by using fingers; adding 2 μ L of recombinant product into 50 μ L of cells, slightly flicking the centrifugal tube wall, mixing, and standing on ice for 30 min;
(2) immediately transferring to ice to cool for 2min after heat shock is carried out for 30s in 42 ℃ water bath; adding 450 μ L of normal temperature non-resistant LB liquid culture medium, and culturing in 37 deg.C shaking table at 250rpm for 1 h;
(3) the cells were centrifuged at 5000rpm for 2min, collected, 100. mu.L of the liquid was resuspended, spread evenly on LB solid plates containing kanamycin resistance, and cultured overnight in a 37 ℃ incubator.
Another object of the present invention is to provide a method for pest resistance of plants or crops using the pest-resistant thifenuron gene, wherein the plant or crop is obtained by introducing a plant expression vector containing a target sequence into cells of the plant or crop, and thereby obtaining transgenic plants or crops and progeny thereof, including seeds and plant or crop tissues of any part, which are resistant or capable of killing pests; or the protein is obtained by homologous or heterologous expression and applied to plants or crops, and the plants or crops obtain insect pest defense capacity.
Wherein, the plants or crops include but are not limited to cotton, corn, wheat, rice and the like.
Another objective of the present invention is to provide a method for detecting the expression of a periiron toxin gene of a transgenic cotton plant, which comprises: taking leaves of a transgenic cotton homozygous strain, extracting total protein of the leaves, and using the total protein for Western Blot detection; the method specifically comprises the following steps:
(1) taking 1-2 g of a fresh sample, and grinding the sample in a mortar by using liquid nitrogen; grinding, placing in a 10mL test tube, adding protein extract, and shaking at 4 deg.C for 30 min;
(2) centrifuging at 12000rpm at 4 deg.C for 20 min; extracting the supernatant, and completing sample preparation;
(3) and taking the supernatant, adding a Loading Buffer, boiling for 5-10 min at 98 ℃, and determining the protein expression condition by using an SDS-polyacrylamide gel electrophoresis method.
Further, the protein extract included 0.01M PBS pH 8, 1mM PMSF, 2mM DTT.
By combining the technical scheme and the technical problem to be solved, the technical scheme to be protected by the invention has the advantages and positive effects that:
aiming at the shortage of available resistance gene resources for crop disease and insect resistant breeding, the invention provides a novel periiron toxin protein gene capable of enabling plants or crops (including but not limited to cotton, corn, wheat, rice and the like) to generate insect resistance, and the invention provides a definite transgenic cotton strain which has Spodoptera frugiperda and cotton bollworm resistance.
The invention provides a novel insect-resistant cyhalothrin gene, which is characterized in that a related plant expression vector is constructed, cotton is transformed by utilizing an agrobacterium-mediated method, and a transgenic cotton strain is obtained; constructing a overexpression vector of the gene; carrying out cotton genetic transformation by adopting an agrobacterium-mediated method; wherein the insect-resistant cyhalothrin gene in the overexpression vector is driven by a 35s promoter. The toxalbumin gene provided by the invention is derived from cycas revoluta and is introduced into cotton, so that the cotton obtains the resistance of spodoptera frugiperda and cotton bollworm, and progeny can be stably inherited.
The invention can further provide or utilize the insect-resistant gene to obtain other transgenic crops and corresponding seeds, and can also transfer the insect-resistant gene into other varieties or similar species with the same background by utilizing a sexual hybridization mode so as to achieve the effect of accelerating the innovation of insect-resistant germplasm of each species.
The invention describes the survival condition of the major agricultural pests such as plutella xylostella, spodoptera frugiperda, cotton bollworm and the like by injecting cycadoxin and transgenic plants, and is characterized as follows:
(1) the diamondback moth of the second day of the third year of consistent age (average body weight 1.33 mg/head) is taken. Each diamondback moth was injected with 2.03 μ g of cyhalothrin, and a control group was set up and injected with an equal volume (207nL) of Phosphate Buffered Saline (PBS). Three replicates were set and 30 larvae were injected per replicate. The mortality rate is counted 24h after injection, and the result shows that the average mortality rate of the diamondback moth is 89.30% after the injection of the cyhalothrin, which is obviously higher than the mortality rate of the control group by 7.78%.
(2) Cotton bollworms aged the second day of the second year (average body weight 2.06 mg/head) were collected at the same age. Each bollworm was injected with 3.38. mu.g of cyhalothrin, and a control group was set up and injected with an equal volume (345nL) of Phosphate Buffered Saline (PBS). Three replicates were set and 30 larvae were injected per replicate. The mortality rate is counted 24h after injection, and the result shows that the average mortality rate of the cotton bollworms is 37.78 percent after the injection of the cyhalothrin and is obviously higher than the mortality rate of a control group by 5.56 percent.
(3) Taking two-instar spodoptera frugiperda (average weight of 1.01 mg/head) with consistent age, uniformly performing a hunger experiment for 8h, and then taking leaves of a transgenic cotton homozygous line as an experimental group, and taking transgenic receptor material 'cotton hundred No. 1' and upland cotton genetic standard line TM-1 as a control group. Three replicates were set, 18 larvae were cultured for each replicate, and mortality was counted for 4 days at 25 ℃. The results show that the average mortality rate of Spodoptera frugiperda is 61.11% after feeding the transgenic cotton for 4 days, which is much higher than the average mortality rate of 24.07% after feeding the cotton "Baimian No. 1" and the average mortality rate of 12.96% after feeding the TM-1.
(4) Two-year-old cotton bollworms (average weight of 3.9 mg/head) with consistent age are taken for feeding experiments of the cotton bollworms. Before the experiment, the starvation experiment is carried out for 8h in a unified way, then the leaves of the homozygous transgenic cotton strain are taken as an experimental group, and the transgenic receptor material 'Bai Cotton No. 1' and the upland cotton genetic standard system TM-1 are taken as a control group. Three replicates were set, 18 larvae were cultured for each replicate, and mortality was counted for 4 days at 25 ℃. The results show that after 4 days of transgenic cotton feeding, the average mortality rate of the bollworms is 59.26 percent, which is far higher than the average mortality rate of 29.63 percent of 'Bai Cotton No. 1' and 14.81 percent of TM-1.
The invention assembles the high-quality genome of cycado and discovers a horizontal gene transfer phenomenon through genome analysis, namely, genes from fungi are found in the cycado genome. The protein coded by the gene is found to contain a cytotoxin structural domain through analysis, and has a pesticidal effect. Furthermore, horizontal gene transfer is a natural transgenic process, and the transferred genes are generally harmless to plants, so the gene is selected as a candidate insect-resistant gene.
The cycadoxin gene is derived from a cycadoxin genome assembled by the inventor, is obtained by analyzing and extracting a gene sequence through the genome, designing a primer and carrying out PCR cloning.
The gene of the cyhalothrin provided by the invention can obviously improve the insect resistance of plants (including but not limited to cotton, corn, wheat, rice and the like), and because the gene is derived from the plants, no toxic effect exists after the gene is transformed into other plants. After the gene is transformed into cotton, the cotton can obtain the resistances of spodoptera frugiperda and cotton bollworm, the insect resistance of the transgenic cotton can be enhanced by utilizing a gene complementation mode, the insect resistance spectrum of the cotton is widened, and the broad-spectrum insect resistance capable of efficiently killing main pests is obtained. The invention successfully clones the cyhalothrin gene, provides the nucleotide sequence and the coding protein amino acid sequence of the gene, and provides theoretical basis and application reference for the creation of plant insect-resistant germplasm and the breeding of new varieties.
The expected income and commercial value after the technical scheme of the invention is converted are as follows: the expected income after the technical scheme of the invention is transformed is related to the approval and popularization area of the subsequent transgenic cotton variety, the insect-resistant cotton area in China is stabilized to about 350 million hectares since 2006, the invention can cultivate the insect-resistant cotton with different backgrounds through the traditional genetic experiment, actively promote to approve the new insect-resistant cotton variety, and the commercial value is very considerable.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the PCR amplification result of the ferritin gene provided in the embodiment of the present invention.
FIG. 2 is a schematic diagram of a plant expression vector constructed according to the present invention, which carries a spectinomycin resistance gene, using a 35S promoter to drive a thifentin gene.
FIG. 3 is a schematic representation of a transgenic cotton plant provided by an embodiment of the present invention.
FIG. 4 is a schematic diagram of PCR detection of the expression of the ferritin gene in leaves of genetically transformed cotton plants according to the present invention, in which the ferritin gene expression in cotton leaves can be clearly seen.
FIG. 5 is a schematic diagram of Western Blot detection of the expression of the ferritin gene in leaves of genetically transformed cotton plants according to the present invention, in which the ferritin gene expression in cotton leaves can be clearly seen.
FIG. 6 is a schematic diagram of insect resistance detection of genetically transformed plant leaves of cotton according to the embodiment of the present invention, in which it can be clearly seen that the cyhalothrin gene significantly inhibits the growth and development of Spodoptera frugiperda and Heliothis armigera.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides an insect-resistant cycotoxin gene, an expression vector and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
First, an embodiment is explained. This section is an explanatory embodiment expanding on the claims so as to fully understand how the present invention is embodied by those skilled in the art.
KOD One according to the invention TM PCR Master Mix was purchased from Toyobo (Shanghai) Biotech Co., Ltd;
Figure BDA0003571196030000071
Quick Gel Extraction Kit、
Figure BDA0003571196030000072
-T1 Cloning Kit、
Figure BDA0003571196030000073
basic Seamless Cloning and Assembly Kit available from Beijing Quanjin Biotechnology Ltd; the plasmid miniprep kit is purchased from OMEGA GmbH; the plant RNA extraction kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd; the RNA reverse transcription kit is purchased from Novozan Biotechnology GmbH; primer synthesis and gene sequencing are completed by Shanghai biological engineering Co., Ltd; gene optimization and synthesis were performed by Henan Shanghai Biotechnology Ltd.
Example 1: cloning of the Perferritin Gene
1. Obtaining of a Perilla Frutescens RNA template
Taking tender leaves of cycas revoluta, and extracting RNA by using a plant RNA extraction kit. Taking about 1g of cycas revoluta tender leaves, quickly freezing the cycas revoluta tender leaves by using liquid nitrogen, putting the cycas revoluta into a mortar, grinding the cycas revoluta into fine powder, and then extracting RNA according to the instruction of a plant RNA extraction kit. RNA concentration was determined using a Nano Drop visible spectrophotometer (Thermo Fisher, USA).
Taking the newly extracted cycad RNA, and carrying out reverse transcription on the RNA into cDNA according to the instructions of a reverse transcription kit.
Using cDNA obtained by reverse transcription as a template, SEQ ID NO. 16: and (3) adding a forward primer: 5'-ATGGTTATGGTAATTGACTATCCAAGTC-3', respectively; reverse primer: 5'-CTATGTCATGCGTAGAATGGCCC-3' is used as a primer to carry out PCR amplification on the cyathrin gene, and the amplification system is as follows:
KOD One TM PCR Master Mix 25μL
upstream primer (10. mu.M each) 1.5. mu.L
Downstream primer (10. mu.M each) 1.5. mu.L
Template 2. mu.L
Sterilized water 20. mu.L
Total volume 50. mu.L
The PCR program is 98 ℃ for 5 min; 30 cycles of 98 ℃ for 30s, 50 ℃ for 30s and 68 ℃ for 3 min; then storing at 68 deg.C for 10min and 4 deg.C. The PCR product was detected by agarose gel electrophoresis (see FIG. 1), followed by detection using
Figure BDA0003571196030000081
The Quick Gel Extraction Kit was purified backCollecting the PCR product, and then using
Figure BDA0003571196030000082
-T1 Cloning Kit PCR product was ligated into T vector, E.coli DH 5. alpha. was transformed using KOD One TM PCR detection is carried out on the PCR Master Mix, and positive clones are selected and sent to Shanghai Biotechnology Limited company for sequencing. Through sequence comparison, the clone with correct sequencing utilizes a plasmid miniprep kit to extract a plasmid, and the plasmid is named as p-T _ Suit, and the sequence information is shown in SEQ ID NO. 1:
atggttatggtaattgactatccaagtctcagagcttcgttatatgccactaagagtgcggtcccatgtcgcccgcttccgggcaatccagagcgggtgtcgcgccgggtatcacgcagaggcgcgggagagatcgggtcccatgcagatttctggatctcccgcgccaaggcatccgccatccggattttcccgcgtcaagagggtccgctccgtgaatcccggagaagattctatcttctccgcgaagcggtgttcaccggccgcaggaccgcctgggccgctaatccagcggcccaacgggcagcatgggaccgcaagagatcccctttcttcggaaggtcttccgatgatattcattcattcagcaagaatccagaagaattcttgactcaaaacactctcagctgcagtttattaactcgcaccgctcagaccgtcacaaaaagcccagtattgcccgcccaaggttgggtcaaagtgggtcgtgtgccaggaagaggagacgctctctttgttgagctggttgccgagaaatcagatgagacagtgaaggcgtacttacttggctacaatggtaagaacgactcccccgagccggcatacgttgatattccaaagaggggcgccgagaacggaacatttttgtttaccgtggatctctcgggctgctccgttgttgtgacacagttggacgagcagacataccgtgttttccacgacaaacgggtgaacagctccattctgtacgacaacgtcgtaatgtatgtggatttcaatcactaccgctatcccagagcccacggacacgggagagatacgggacaagcggtcgtctgtatgcaattcgccgacggagtgtggaaattgtttctccagcggcacgagacggcgaattctctcccaaagatatggtcccgtggcgatggcaaaccggagattttcagcgacgccgaaccccgtccacatatggaagatggcataaaggaatttctcaatcggcgtcgagctttgcaagacgaattgaagcacttcgccaacacgcttgaagtggacccggacatagtgtccaacgctgtcgacggcgaatacgaaggcggccagttttccaggaacgacgatgcaattcgtggatggaacgaattacgtgcaagcatgagacagaaaatggctgaaaggaagaaactgttgcaaacaaagaaaggtatgctagactcattcaacaacccggatcaccccctctataaatttcgcaaacaagtgctcgacgagctcaaggaaatgagtgcatacgacaacaccttggaggcatccgagattgcggaccgtacttggctgtggttggagatcaaaaaagccaaaggtacgaatgcagtggtaaacagtggagcagccccgaagcagccgagtcttaccgttggtgaacgctacgcagagatggaggcggccctttggatgcgtagcgacacagcggcgtttgagcaagggtatgaatcttatcaaaccgttcgcgtgccgggttacaatgaagatatgacatccattgcaatgaagaagttgttcgttgagagcagtctgactgacgagcagctgggcgccttgtgcaagcgtatatccattgctagcgatcaagagaaccatcttctgacacagaagacgcaggagattgagagcactttcaaacaattttcagatggcagctttttggagtcggcgcctcaggatttcttcctgagacgagtcggtgataaatctggaggacgctgctatcctcttgtaagggtaatggccgtcgcactgtctgaaggagaagacgttgcccagaattttatggagaacctgaggacattgtctaggaatccagagtattcaggcgaatcggcaagttcacaagccacaaatgcagggttgttcagagatgccctggccggcctgcacgcgaatgtggaagccattgccgcatctgagaaactacctgctgtgtttggactgcccgatgttgctacaatgttgagccaagcgagcgaaagcaaggtttacgctttgaataccgaaacacacagcatgctggttgcggtgattgtagacaaacagagtctggcgcgcaagtattacttctatgatcccaactttctcatggccggtttctccgacataactcgtttctcggaagccctcaagtatcactttgtgaatctccagttcgccggaaactacaaagcgtttggcacagcggaggctccccagtttaacctggtccttattgatacgaccgccatggaggatgcgcatgttgacggagggatcaccgtgaaacaattgagcgagggcgttgatcttactaatgtctttgacagcaccaaaaaggctcgtgtggtaactgaggcagcagctcgcttggctgaagaccaacaactgcgcactggcctcaccataatagaggctgaccagtgggcaaacaggtggcaagatgccgtcatacgattgaaactcaaggaaggattaagtggagagtggatgccattgctggagaaaatgcaagaaaacgcagatggttcttatcagattgatttcatcaacaccaagaatactggggaaatccgtggggtttccactagagatgcgacgttcaaagaattcaaatcctacctggataaaaatttagacgtattgcataaggatttcatctttcaggatggagttttcaaaccactggagggagagcccattgacgggttgaatgcaatgttcgcggtccaaatgattatagattggttcaaccgggggactaatgctgaggaagatgcggcctcaggatccaccaatgcccaactttccctagcgattaaagtgcacagctacattaacatggcccaagtagcgcatactgcagttacagatgttgccaaggtatacaaccttgtaaagtcagcactgtatggcgagcaggtagctggggagacatcgctgttcacatttgggaaggcgttgaaatttgcagcaacagaagggttgggagtcttactaggggcgtcctttgtgatcttggatgcctaccagctgtccctcgtggaaaacgatgtgcagaaggccattatcggcacacaactagcctttgattccgcaagctttctagcgggtattggtgcaattgttgctggagcatcgggcgtgggcatgattttgggcggagctggcgtggtattgggaggcctgggagttggctttggagccttagctcaggcattcggggaaatcacagcagacgcccaactggtgggcaagtacttcgcactcttggacgaggcataccgcggtggcggatacagatacgaatctaaagacaaggcattgatgcctcttgcaggagcagtcatcgacaaaatacgactggccgacaaacatctccaagttcatttcgacagccaattcatatatcgaacccatcatggcagcacaggatccggcaaaattaattacttcttctgggctggcgacatgccgcagatggtccacgatcgctcccaagctatcaatgttcgagagcaaattgggtataagaacgataatgctactttggagctaccggaggatgcctctgatctgatcctgcctgccacgcccaagtcatttatcacttaccgatacgagattctaccattcgctacttcccgccacgatacgggtttcgatgtcattagacgcctggaagaagactacagattcgattacgatttctacatcttcccatctgaatacacaatcagaaccatcaaacacgagtacgtagaaacaaaggtggcagtagtcctggataggcgctctttgcgactgggagcacctgagcttctcaaggagcaacatggctatttgatctatgagatcgaggggaccggaggacaatacgttatatgtcttaaccagggatccactgtcacactgaagaccaccggatcagaggcaaataaaacattatggatacttgatgcccgacaactcattgcaggcaacgattcaatcacggtctctcaagatagcatcaccatcggcagcacaacagtccatctccgtaaccaagatttcgattgcctacagatcgtcatgagtcgggaggagcttctccaagtggacttggcgtctggagaagcccgcgtgttttcagaagatgccagtaaatggctcgacaccaaagaaacaatccatgaacatctccataagttggctgaagaacacaggttgcatggccagtttgtagtggtgaacaattattatcgtggagtgggccgtgcctactatgatgctgctcacgatcggatgttgtatacagatgtggaagatgaaactctgactcgggatgctgttcttgccggcgttacaggcgaagatgcctacttctacaatgtaaagcagaatgccctgtggcgagtgaatgcaggcacacaccaagtaataaccaaatactatccgacgttactggaggaccctgacagcaacatggcaagagttgagcagcatggcaacatcgtcgtgatggccatgagcaggctcaaccacccggacaagggagtaaatggaatcatctatctcgttcatcaagaaagcatcgtgcttactgctgtgatcggcaattcagagatgctcaaacgtcttcgccaaacgagtacaattagcttaacagaattgcgaggtgtcaatgacatggctctggtttccacgatggacagattacccggccaaaaggttgtcgctaccaacgctgagtttgtgacacttctggctaaggatgagcaaggagccaatatcaaggagcagacggtctattgccaacaaggtacgggccagaccaacgatacatctattgaaagcatcccgattccagggctctctaatcttttaaccgtgtacggtgaagtattcgctacaactaatgatggtcgcatccttcgcctgcttggggacggctcaacatgtctcgagggcatcaatgagcattggctcatcaagcatccaaaatggtggcaggacttggctaccttggaggcagagaaagccacgacagttgcagtattcggtgttagagacctacaaaatgcagtggtgccaatttggtaccacaacggaaagatcgcggtggcctcatcaaaactgtatggcaaacaactgcaatttttggggtttaacagcaacggtgaagcttggttgttcgacttggacgacggcattctgtatagccagccgcttgtggatagctttctgtatgcaatctttagtttggacgggaagcttattgcaccaatctttattccacaagtggagcaaatgctcagcggccagcatctcagaaaccctgtcatcgtggacggccgtttgcatgtgactaccatagacggtgttgttgttggatttgacaagaagtcgccaactctggttgcagtgaacaaggcctggcagatatctcgtcagaaccgattagaagaggccctcggaaacctgctgtccaattggtctgatcacaatgatgcgattgtgttgcagggtgatccagaatcagcgccggcatggtattatgttccatcaagagaaatagtgaccgccaatggtctaacatgggcggatcatcctctatggctaggaatggctgtcggtgctgcaacgccatacatctacagcacatcccgacgccgtgtgtatgttgtggagagcaaaaacatggccaaagacaaaggagaattcgcagcagcacattgctttgcaaaatcacttgtgctacagagcatcgttcccgaggaaataacgattccgacgctggctggcatcgatttcgcactcgttgcagcgacaaatgcaaccattgtaatagaccgagacaattggaagcattacaaggccattgtgctcgacaacaaggtggatcaggacggtggcgctaaggtggtagtcggcagtgagattcccatgaaagttctggtgcgcaaatttcagggaaatctggagttcttcgatccaaacaatggtaatgttgttatactccagaatgtgttgaagggagacggagcttgttgccagatagagatcaaagtaggcgatgctgagactatgcttgtatccgatttctcggagatattctcaaagacgcatcctctggtggcaagccaagtatttgagcttggggatgtgattgcagtcacaaaacacaaatcggactatgcggatctgtgggccattctacgcatgacatag
the invention provides a homologous gene of the insect-resistant cycadoxin gene in cycads, and the nucleotide sequence of the homologous gene is as follows:
SEQ ID NO.2：
ATGGAGGAGGCTGACATGGAGAACCGTGGATACGAAGAACGGCACTCACAGTCACAGTCTGCTACACCGCTCCGCCGAAGGAAGAGATCCCCTTTCTTCGGAAGGTCTTCCGATGATATTCATTCATTCAGCAAGAATCCAGAAGAATTCTTGACTAAAAACACTCTCAGCTGCAGTTTATTAACTCGCACCGCTCAGACCGTCACAAAAAGCCCAGTATTGCCCGCCCAAGGTTGGGTCAAAGTGGGTCGTGTGCCAGGAAGAGGAGACGCTCTCTCGGGCTGCTCCGTTGTTGTGACACAGTTGGACGAGCAGACATACCGTGTTTTCCACGACAAACGGGTGAACAGCTCCATTCTGTACGACAACGTCGTAATGTATGTGGATTTCAATCACTACCGCTATCCCAGAGCCCACGGACACGGGAGAGATACGGGACAAGCGGTCGTCTGTATGCAATTCGCCGACGGAGTGTGGAAATTGTTTCTCCAGCGGCACGAGACGGCGAATTCTCTCCCAAAGATATGGTCCCGTGGCGATGGCAAACCGGAGATTTTCAGCGACGCCGAACCCCGTCCACATATGGAAGACGGCATAAAGGAATTTCTCAATCGGCGTCGAGCTTTGCAAGACGAATTGAAGCACTTCGCCAACACGCTTGAAGTGGACCCGGAGATAGTGTCCAACGCTGTCGACGGCGAATACGAAGGCGGCCAGTTTTCCAGGAACGATGATGCAATTCGTGGATGGAACGAATTACGTGCAAGCATGAGACAGAAAATGGCTGAAAGGAAGAAACGGTTGGAAAGAAACAAAGGTATGCTAGACTCATTCAACAACCCGGATCACCCCCTCTATAAATTTCGCAAACAAGTGCTCGACGAGCTCAAGGAAATGAGTGCATACGACAACACCTTGGAGGCATCCGAGATTGCGGACCGTACTTGGCTGTGGTTGGAGATCAAAAAAGCCAAAGGTACGAATGCGGTGGTAAACAGTGGAGCAGCCCCGAAGCAGCCGAGTCTTACCGTTGGTGAACGCTACGCAGAGATGGAGGCGGCCCTTTGGATGCGTAGCGACACAGCGGCGTTTGAGCAAGGGTATGAATCTTATCAAACCCTTCGCGTGCCGGGTTACAATGAAGATATGACATCCATTGCAATGAAGAAGTTGTTCGTTGAGAGCAGTCTGACTGACGAGCAGCTGGGCGCCTTGTGCAAGCGTATATCCATCGCTAGCGATCAAGAGAACCATCTTCTGACACAGAAGACGCAGGAGATTGAGAGCACTTTCAAACAATTTTCAGATGGCAGCTTTTTGGAGTCGGCGCCTCAGGATTTCTTCCTGAGACGAGTCGGTGATAAATCTGGAGGACGCTGCTATCCTCTTGTAAGGGTAATGGCCGTCGCACTGTCTCAAGGAGAAGACGTTGCCCAGAATTTTATGGAGAACCTGAGGACATTGTCTAGGAATCCAGAGTATTCAGGCGAATCGGCAAGTTCACAAGCCACAAATGCAGGGTTGTTCAGAGATGCCCTTGCCGGCCTGCACGCGAATGTGGAAGCCATCGCCGCATCTGAGAAACTACCTGCTGTGTTTGGACTGCCCGATGTTGCTACAATGTTGAGCCAAGCGAGCGAAAGCAAGGTTTACGCTTTGAATACCGAAACACACAGCATGCTGGTTGCGGTGATTGTAGACAAACAGAGTCTGGCGCGCAAGTATTACTTCTATGATCCCAACTTTCTCATTGCCGGTTTCTCCGACATAACTCGTTTCTCGGAAGCCCTCAAGTATCACTTTGTGAATCTCCAGTTCGCCGGAAACTACAAAGCGTTTGGCACAGCGGAGGCTCCCCAGTTTAACCTGGTCCTTATTGATACGACCGCCATGGCGGATGCGCATGTTGACGGAGGGATCACGGTGAAACAATTAAGCGAGGGCGTTGATCTTACTAATGTCTTTGACAGCACCAAAAAGGCTCGTGTGGTAACTGAGACAGCAGCTCGCTTGGCTGAAGACCAACAACTGCGCACTGGCCTCACCATAATAGAGGCTGACCAGTGGGCAAACAGGTGGCAAGATGCTGTCATACGATTGAAACTCAAGGAAGGATTAAGTGGAGAGTGGATGCCATTGTTGGAGAAAATGCAAGAAAACGCAGATGGTTCTTATCAGATTGACTTCATCAACACCAAGAATACTGGGGAAATCCGTGGGGTTTCCACTAGAGATGCGACGTTCAAAGAATTCAAATCCTACCTGGATAAAAATTTAGACGTATTGCATAAGGATTTCATCTTTCAGGATGGAGTTTTCAAACCACTGGAGGGAGAGCCCATTGACGGGTTGAATGCAATGTTCGCTGTCCAAATGATTATAGATTGGTTCAACCGGGGGACTAATGCTGAGGAAGATGCGGCCTCAGGATCCACCAATGCCCAACTTTCCCTAGCGATTAAAGTGCACAGCTACATTAACATGGCCCAAGTAGCGCATACTGCAGTTACAGATGTTGCCAAGGTATACAATCTTGTAAAGTCAGCACTGTATGGCGAGCAGGTAGCTGGGGAGGCATCGCTGTTAACATTTGGGAAGGCGTTGAAATTTGCAGCAACAGAAGGGTTGGGAGTCTTACTAGGGGCGTCCTTTGTGATCTTGGATGCCTACCAGCTGTCCCTCGTGGAAAACGATGTGCAGAAGGCCATTATCGGCACACAACTAGCCTTTGATTCCGCAAGCTTTCTAGCGGGTATTGGTGCAATTGTTGCTGGAGCATCGGGCGTGGGCATGATTTTGGGCGGAGCTGGCGTGGTATTGGGAGGCCTGGGAGTTGGCTTTGGAGCCTTAGCTCAGGCATTCGGGGAAATCACAGCAGATGCCCAACTGGTGGGCAAGTACTTCGCACTCTTGGACGAGGCATACCGCGGTGGCGGATACAGATACGAATCTAAAGACAAGGCATTGATGCCTCTTGCAGGAGCAGTCATCGACAAAATACGACTGGCCGACAAACATCTCCAAGTTCATTTCGATAGCCAATTCATATATCGGACCCATCATGGCCTCACAGGATCCGGCAAAATTAATTACTTCTTCTGGGCTGGCGACATGCCGCAGATGGTCCACGATCGCTCCCAAGCTATCAATATTCGAGAGCAAATTGGATACAAGAACGATCATGCTACTTTGGAGCTACCGGAGGATGCCTCTGATCTGATCCTGCCTGCCACGCCCAAGTCATTTATCACTTACCAATACCAGATTCTACCATTTGCTACTTCCCGCCACGATAGGGGTTTCGATGTCATCAGACGCCTGGAAGAAGACTACAGATTCGATTACGATTTCTACATCTTCCCATCTGAATACACAATTAGAACCATCAAACACGAGTACGTAGAAACAAAGGTGGCAGTAGTCCTGGATAGGCGCTCTTTGCGACTGGGAGCACCTGAGCTTCTCAAGGAGCAACATGGCTATTTGATCTATGAGATCGAGGGGACCGGAGGACAATACGTTATATGTCTTAACCAGGGATCCACTGTCACACTGAAGACCACCGGATCAGAGGCAAATAAAACATTATGGATACTTGATGCCCGGCAACTCATTGCAGGCAACGATTCAATCACGGTCTCTCAAGATAGCATCACCATCGGCAGCACAACAGTCCATCTCCGTAACCAAGATTTCGATTGCCTACAGATCGTCATGAGTCGGGAGGAGCTTCTCCAAGTGGACTTGGCGTCTGGAGAAGCCCGCGTGTTTTCAGAAGATGCCAGTAAATGGCTCGACACCAAAGAAACAATCCATGAACATCTCCATAAGTTGGCTGAAGAACACAGGTTGCATGGCCAGTTTGTAGTGGTGAACAATTATTATCGTGGAGTGGGCCGTGCCTACTATGATGCTGCTCACGATCGGATGTTGTATACAGATGTGGAAGATGAAACTCTGACTCGGGATGCTGTTCTTGCCGGCGTTACAGGCGAAGATGCCTACTTCTACAATGTAAAGCAGAATGCCCTGTGGCGAGTGAATGCAGGCACACACCAAGTAATAACCAAATACTATCCGACGTTACTGGAGGACCCTGACAGCAAAATGGCAAGAGTTGAGCAGCATGGCAACATCGTCGTGATGGCCATGAGCAGGCTCAACCACCCCGACAAGGGAGTAAATGGAATCATCTATCTCGTTCATCAAGAAAGCATGGTGCTTACTGCTGTCATCGGCAATTCAGAGATGCTCAAACGTCTTCGCCAAACGAGTACGATTAGCCTAACAGAATTGCGAGGTGTCAATGACATGGCTCTGGTTTCCACGATGGACAAATTACCCGGCCAGAAGGTTGTCGCCACCAATGCTGAGTTTGTTACTATTCTGGCTAAGGATGAGCAAGGGAGCCAAGTATGTTTTTGGCTGCGCACCGTGGATGGAGTTCTAATCAAGCCGAGCATCCAGCCACCCATACCAACGGACCTAACCTTGACTGTTTCATCCCACAAAGTGGCTGGACAGGATGTATTCTACTTTTACAGTATCAAGGAGCAGACGGTCTATTGCCAACAAGGGACGGGCCAGACCAACGATACATCTATTGAAAGCATCCCGATTCCAGGGCTCTCTAATCTTTTAACCGTGTACGGTGAAGTATTCGCTACAACTGATGATGGTCGCATCCTTCGCCTGCTTGGGGATGGCTCAACATATCTTGACGGCATCAATGAGCATTGGCTCATCAAGCATCCAAAATGGTGGCAGGACTTGGCTACCTTGGAGGCAGAGAAAGCCACGACAGTTGCAGTATTCGGTGTTAGAGACCTACAAAATGCAGTGGTGCCAATTTGGTACCACAATGGAAAGATCGCGGTGGCCTCATCAAAACTGTATGGCAAACAACTGCAATTTTTGGGGTTTAACAGCAACGGTGAAGCTTGGTTGTTCGACTTGGACGACGGCATTCTGTATAGCCAGCCGCTTGTGGATAGCTTTCTGTATGCAATCTTTAGTTTGGACGGGAAGCTTATTGCACCAATCTTTATTCCACAAGTGGAGCAAATGCTCAGCGGCCAGCATCTCAGAAACCCTGTCATCGTGGACGGCCGTTTGCATGTGACTACCATAGACGGTGTTGTTGTTGGATTTGACAAGAAGTCGCCAACTCTGGTTGCAGTGAACAAGGCCTGGCAGATATCTCGTCAAAACCGATTAGAAGAGGCCCTCGGAAACCTGCTGTCCAATTGGTCTGATCACAATGATGCGATTGTGTTGCAGGGTGATCCAGAATCAGCGCCGGCATGGTATTATGTTCCATCAAGAGAAATAGTGACCGCCAATGGTCTAACATGGGCGGATCATCCTCTATGGCTTGGAATGGCTGTCGGTGCTGCAACGCCATACATCTACAGCACATCCCGACGCCGTGTGTATGCTGTGGAGAGCAAACACATGGCCAAAGACAAAGGAGAATTCGCAGCAGCACATTGCTTTGCAAAATCACTTGTGCTACAGAGCATCGTTCCCGAGGAAATAACGATTCCAACGCTGGCAGGCATCGATTTCGCACTCGTTGCAGCGACAAATGCAACCATTGTAATACACCGAGACAATTGGAAGCACTACAAGGCCATTGTGCTCGACAACAAGGTGGATCAGGACGGTGGCGCTAAGGTGGTAGTCGGCAGTGGGATTCCCATGAAAGTTCTGGTGCGCAAATTTCAGGGAAATCTGGAGTTCTTCGATCCAAACAATGGTAATGTTGTTATACTCCGGAATGTGTTGAAGGGAGACGGAGCTTGTTCCCAGATAGAGATCAAAGTAGGCGATGCTGAGACTATGCTTGTATCCGATTTCTCGGAGATATTCTCAAAGACGCATCCTCTGGTGGCAAGCCAAGTATTTGAGCTTGGGGATGTGATTGCAGTCACAAAACACAAATCGGACTATGCGGATCTGTGGGCCATTCTACGCATGACATAG
SEQ ID NO.3：
ATGGCCGTCGCACTGTCTCAAGGAGAAGACGTTGCCCAGAATTTTATGGAGAACCTGAGGACATTGTCTAGGAATCCAGAGTATTCAGGCGAATCGGCAAGTTCACAAGCCACAAATGCAGGGTTGTTCAGAGATGCCCTTGCCGGCCTACACGCGAATGTGGAAGCCATCGCCGCATCTGAGAAACTACCTGCTGTGTTTGGACTGCCCGATGTTGCTACAATGTTGAGCCAAGCGAGCGAAAGCAAGGTTTACGCTTTGAATACCGAAACACACAGCATGCTGGCTGCGGTGATTGTAGACAAACAGAGTCTGGCGCGCAAGTATTACTTCTATGATCCCAACTTTCTCATGGCCGGTTTCTCCGACATAACTCGTTTCTCAGAAGCCCTCAAGTATCACTTTGTGAATCTCCAGTTCGCCGGAAACTACAAAGCGTTTGGCACAGCGGAGGCTCCCCAGTTTAACCTGGTCCTTATTGATACGACCGCCATGGCGGATGCGCATGTTGACGGAGGGATCACCGTGAAACAATTGAGCGAGGGGGTTGATCTTACTAATGTCTTTGACAGCACCAAAAAGGCTCGTGTGGTAACTGAGGCAGCAGCTCGCTTGGCTGAAGACCAACAACTGCGCACTGGCCTCACCATAATAGAGGCTGACCAGTGGGCAAACCGGTGGCAAGATGCCGTCATACGATTGAAACTCAAGGAAGGATTAAGTGGAGAGTGGATGCCATTGCTGGAAAGAATGCAAGAAAACGGACATGGTTCATATCAGATTGAATTCCTCAACACCAAGAATACTGGGGAAACCCGTTGGGTTTCCACTAGAGATGCGACGTTCAAAGAATTCAAATCCTACCTGGATAAAAATTTTGACGCATTGCGTAAGGATTTCATCTTTCAGGATGGAGTTTTCAAACCACTGGATGGAGAGCCCATTGACGGGTTGAATGCAATGTTCGCGGTCCAAATGATTATAGATTGGTTCAACCCGGGGACTAATGCTGAGGAAGATGCGGCCTCAGGATCCACCAATGCCCAACTTTCCCTAGCGATTAAAGTGCACAGCTACATTAACATGGCCCAAGTAGCGCATACTGCAGTTACAGATGTTGCCAAGGTATACAATCTTGTAAAGTCAGCACTGTATGGCGAGCAGGTAGCTGGGGAGGCATCGCTGTTAACATTTGGGAAGGCGTTGAAATTTGCAGCAACAGAAGGGTTGGGAGTCTTACTAGGGGCGTCCTTTGTGATCTTGGATGCCTACCAGCTGTCCCTCGTGGAAAACGATGTGCAGAAGGCCATTATCGGCACACAACTAGCCTTTGATTCCGCAAGCTTTCTAGCGGGTATTGGTGCAATTGTTGCTGGAGCATCGGGCGTGGGCATGATTTTGGGCGGAGCTGGCGTAATATTGGGAGGCCTGGGAGTTGGCTTTGGAGCCTTAGCTCAGGCATTCGGGCAAATCACAGCAGATGCCCAACTGGTGGGCGAGTACTTCGCACTCTTGGACGAAGCATACAGCGGTGGCGGATACAGATACGAATCCAAAGACAAGGCATTGATGCCTCTTGCAGGAGCAGTCATCGACAAAATACGACTGGCCGACAAACGTCTCCAAGTTCATTTCGACAGTCAATTCATATATCGAACCCATCATGGCAGCACAGGATCCGGCAAAATTAACTACTTCTTCTGGGTTGGCGACATGCCGCAGATGGTCCACGATCGCTCCCAAGCTATCAATGTTCGAGAGCAAATAGGGTATAAGAACGATAGTGCTACTTTGGAGCTACCGGAGGATGCCTCCGATCTGATCCTGCCTGCCACGCCCAAGTCATTTATCACTTACCGATACGAGATTCTACCATTTGCTACTTCCCGCCACGATAGGGGTTTCGATGTCATTAGACGCCTGGAAGAAGACTACAGATTCGATTACGATTTCTACATCTTCCCATCTGAATACACAATCAGAACCATCAAACACGAGTACGTAGAAACAAAGGTGGCAGTAGTCCTGGATAGGCGCTCTTTGCGACTGGGAGCACCTGAGCTTCTCAAGGAGCAACATGGCTATTTGATCTATGAGATCGAGGGGACCGGAGGACAATACGTTATATGTCTTAACCACGGATCCACTGTCACACTGAAGACCACCGGATCAGAGGCGAATAAAACATTATGGATACTTGATGCCCGGCAACTCATTGCAGGCAACGATTCAATCACGGTCTCTCAAGATAGCATCACCATCGGCAGCACAACAGTCCATCTCCGTAACCAAGATTTCGATTGCCTGCAGATCGTCATGAGTCGGGACGAGCTTCTCCAAGTGGACTTGGCGTCTCGAGAAGTCCGCGTGTTTTCAGAAGATGCCAGTAAATGGCTCGACACCAAAGAAACAATCCATGAACATCTCCATAAGTTGGCTGGAGAACACAGGTTGCATGGCCAGTTTGTAGTGGTGAACAATTATTATCGTGGAGTGGGCCGTGCCTACTATGATGCTGCTCACGATCGGATGTTGTATACAGATGTGGAAGATGAGACTCTGACTCGGGATGCTGTTCTTGCCGGCGTTACAGGCGAAGATGCCTACTTCTACAATGTAAATCAGAATGCCCTGTGGCGAGTGAATGCAGACACACACCAAGTAATAACCAAATACTATCCGACGCTCCTGGAGGACCCTGACAGTAAAATGGCAAGAGTCGAGCAGCATGGCAACATCGTCGTGATGGCCATGAGCAGGCTCAACCACCCCGACAAGGGAGTAAATGGAATCATCTATCTCGTTCACCAAGAAAGTATGGTGCTCACTGCTGTGATCGGCAATTCAGAGATGCTCAAACGTCTTCGCCAAACGAGCACGATTAGCTTAACAGAATTGCGAGGCGTCAATGACATGGCCCTGGTTTCCACGATGGACAGATTACCCGGCCAAAAGGTTGTCGCCACCAACGCTGAGTTTGTGACACTTCTGGCTAAGGATGAGCAAGGGAGCCAAGTCTGTTTTTGGCTGCGCACCGTGGATGGAGTTCTAATCAAGCCGAGCATCCAGCCACCCATACCAACGGACCTAACCTTGACTGTTTCATCCCACAAAGTGGCTGGACAGGATGTATTCTACTTTTACAGTATCAAGGAGCAGACGGTCTATTGCCAACAAGGTACGGGCCAGACCAACGATACATCTATTGAAAGCATCCCGATTCCAGGGCTCTCTAATCTTTTAACCGTGTACGGTGAAGTATTCGCTACAACTAATGATGGTCGCATCCTTCGCCTGCTTGGGGACGGCTCAACATATCTTGAGGGCATCAATGAGCATTGGCTCATCAAGCATCCAAAATGGTGGCAGGACTTGGCTACCTTGGAGGCAGAGAAAGCCACGACAGTTGCAGTATTCGGTGTTAGAGACCCACAAAATGCAGTGGTGCCAATTTGGTACCACAATGGAAAGATCGCAGTGGCCTCATCAAAACTGTATGGCAAACAACTGCAATTTTTGGGGTTTAACAGCAACGGTGAAGCTTGGTTGTTCGACTTGGACGACGGCATTCTGTATAGCCAGCCGCTTGTGGATAGCCAAGCTGTGGATGCAATCTTTAGTTTGGACGGGAAGCTTATTGCACCAAACCTTATTCCACAAGTGGAGCAAATGCTCAGCGGCCAGCATCTCAGAAACCCTGTCATCGTGGACGGCCGCTTGCATGTGACTACCATAGACGGTGTTGTTGTTGGATTTGACAAGAAGTCGCCAACTCTGGTTGCAGTGAACAAAGCCTGGCAGATATCTCGTCAGAACCGATTAGAAGAGGCCCTCGGAAACCTGCTGTCCAATTGGTCTGATCACAATGATGCGATTGTGTTGCAGGGTGATCCAGAATCAGCACCGGCATGGTATTATGTTCCATCAAGGGAAATAGTGACCGCCAATGGTCTAACATGGGCGGATCATCCTCTATGGCTAGGAATGGCTGTCGGTGCTGCAACGCTATACATCTACAGCACATCTCGACGCCGTGTGTATGCTGTGGAGAGCAAAAAGATGGCCAAAGACAAAGGAGAATTCGCAGCAGCACATTGCTTTGCAAAATCACTTGTGCTACAGAGCATCGTTCCCGAGGAAATAACGATTCCAACGCTGGCTGGCACCGATTTCGCACTCGTTGCAGCGACAAATGCAACCATTGTAATAGACCGAGACAATTGGAAGCACTACAAGGCCATTGTGCTCGACAACAAGGTGGATCAGGACGGTGGCGCTAAGGTGGTAGTCGGCAGTGAGATTCCCATGAAAGTTCTGGTGCGCAAATTTCAGGGAAATCTGGAGTTCTTCGATCCAAACAATGGTAATGTTGTTATACTCCAGAATGTGTTGAAGGGAGACGGAGCTTGTTCCAAGATAGAGATCAAAGTAGGCGATGCTGAGACTATGCTTGTATCCGATTTCTCGGAGATATTCTCAAAGACGCATCCTCTGGTGGCAAGCCAAGTATTTGAGCTTGGGGATGTGATTGCAGTCACAAAACACAAATCGGACTATGCGGATCTGTGGGCCATTCTACGCATGACATAG
SEQ ID NO.4：
ATGGAGGAAGTTGACATGCAAAACCGTGGATACGAAGAATGGCACTCACAGTCACAGTCTGCTACAACGCTCCGCCGAAGGAAGAGATCCCCTGTCTTCGGAAGGTCTTCCGATGACATTCATTCGTTCAGCAAGAATCCAGAAGAATTCTTGACTCAAAACACTCTAAGCTGCAGTTTATTAACTCGCACCGCTGAGACCGTCACAAAAAGCCCAGTATTGCCCGAACGAGGTTGGGTTAAAGTGGGTCGTGTGCCAGGAAGAGGAGACGCTCTCTTTGTTCAAGTGGTTGCCGAGCAATCATATGACACCGTGAAAGCGTACTTACTTGGCTACAATGGTGAGAACGACTCCCCCAACCCGGCATATGTTGATATTCCAAAGAGGGCCGCCGAGAACGGAACATTTTTGTTTACCGTTGATCTCTCGGGCTGCTCCGTTGTTGTGACGCAGTTGGACGAGCAGACATACCGTGTTTTCCACGACAAACGGGTGAACAGCTCCATTCTGTACGACAACGTCGTAATGTATGTGGATTTCAATCACTACCGCTATCCCAGAGCCCATGGACACGGGAGAGATACCGGACAAGCGGTCGTCTGTATGCAATTCGCCGACGGAGTGTGGAAATTGTTTCTCCAGCGGCACGAGACGGCGAATTCTCTCCCAAAGATATGGTCCCGTGGCGATGGCAAACCGGAGATTTTCAGCGACGCCGAACCCCGTCAACATATGGAAGACGGCATAAAGGAATTTCTCAATCGGCGTCGAGCTTTGCAAGACGAGTTGAAGAAATTCGCCGACATGTTTGGAGTGGACCCGGAGATAGTGTCCAACGCTGTCGACGGCAAATACAAGGGTGGCCAGTTTTCCAGGAACGATGATGCAATTCGTGGATGGAACGAATTACGTGCAAGCATGAGACAGAAAATGGCCGAAAGGAACAAAGTGTTGCTAACAATGAAACTGGAGCAGCCCCGAAGCAGCCGAGTCTTACCGTTGGTGAACGCTACGCAGAGATGGAGGCGGCCCTTTGGATGCGTAGCGACACAGCGGCGTTTGAGCAAGGCTGGGCGCCTTGTGCAAGCGTATATCCATCGCGATCAAGAGAACCATCTTCTGACACAGAAGACGCAGGAGATTGAGAGCACTTTCAAACAATTTTCAGATGGCAGCTTTTTGGAGTCGGCGCCTCAGGATTTCTTCCTGAGACGAGTCGGTGATAAATCTGGAGGACGCTGCTATCCTCTTGTAAGGGTAATGGCCGTCGCACTGTCTCAAGGAGAAGACGTTGCCCAGAATTTTATGGAGAACCTGAGGACATTGTCTAAGAATCCAGAGTATTCAGGCGAATCGGCAAGTTCACAAGCCACAAATGCAGGGTTGTTCAGAGATGCCCTTGCCGGCCTGCACGCGAATGTGGAAGCCATCGCCGCATCTGAGAAACTACCTGCTGTGTTTGGACTGCCCGATGTTGCTACAATGTTGAGCCAAGCGAGCGAAAGCAAGGTTTACGCTTTGAATACCGAAACACACAGCATGCTGGCTGCGGTGATTGTAGACAAACAGAGTCTGGCGCGCAAGTATTACTTCTATGATCCCAACTTTCTCATGGCCGGTTTCTCCGACATAACTCGTTTCTCAGAAGCCCTCAAGTATCACTTTGTGAATCTCCAGTTCGCCGGAAACTACAAACCGTTTGGCACAGCGGAGGCTCCCCAGTTTAACCTGGTCCTTATTGATACGACCGTCATGGCGGATGCGCATGTTGACGGAGGGATCACCGTGAAACAATTGAGCGAGGGGGTTGATCTTACTAATGTCTTCGACAGCACCAAAAAGGCTCGTGTGGTAACTGAGGCAGCAGCTCGCTTGGCTGAAGACCAACAACTGCGCACTGGCCTCACCATAATAGAGGCTGACCAGTGGGCAAACCGGTGGCAAGATGCCGTCATACGATTGAAACTCAAGGAAGGATTAAGTGGAGAGTGGATGCCATTGCTGGAAAAAATGCAAGAAAACGCAGATGGTTCTTATCAGATTGACTTCATCAACACCAAGAATACTGGGGAAATCCGTGGGGTTTCCACTAGAGATGGGACGTTCAAAGAATTAAAATCCTACCTGGATAAAAATTTAGACGCATTGCATAAGGATTTCATCTTTCAGGATGGAGTTTTCAAACCACTGGAGGGAGAGCCCATTGACGGGTTGAATGCAATGTTCGCAGTCCAAATGATTATAGATTGGTTCAATCGGGGGACTAATGCTGAGGAAGATGCGGCCTCAGGATCCACCAATGCCCAACTTTCCCTAGCGATTAAAGTGCACAGCTACATTAACATGGCCCAAGTAGCGCATACTGCAGTTACAGATGTTGCCAAGGTATACAGCCTTGTGAAGTCAGCACTGTATGGCGAGCAGGTAGCTGGGGGGGCATCGCTGTTAACATTTGGGAAGGCGCTGAAACTTGCAGCAACAGAAGGGTTGGGAGTCTTACTAGGGGCGCCTGTTGTGATCTTGGATGCCTACCAGCTGTCCCTCGTGGAAAACGATGTGCAGAAGGCCATTATTGGCACACAACTAGACTTTGATTCCGCAAGCTTTTTAGTGAGTATCGGGGCAGCAGTTGCTGGAGTATCGGGCGTGGGCATCGTTTTGAGCGGAGCTGGCGTGTTATTGGCAGGCCTGGGAGTTGGCTTTGGAGCCTTAGCTCAGGCATTCGGGGAAATCACAGCAGACGCCCAGCTGGTGGGCAAGTACTTCGCACTCTTGGACGAGGCATACCGCGGTGGCGGATACAGATACGAATCTAAAGACAAGGCATTGATGCCTCTTGCAGGAGCAGTCATCGACAAAATACGACTGGCCGACAAACATCTCCAAGTTCATTTCGACAGCCAATTCATATATCGAACCCATCATGGCAGCACAGGATCCGGCAAAATTAATTACTTCTTCTGGGTTGGCGACATGCCGAAGATGGTCCACGATCGCTCCCAAGCTATCAATGTTCGAGAGCAAATTGGGTATAAGAACGATAGTGCTACTTTGGAGCTACCGGAGGATGCCTCTGATCTGATCCTGCCTGCCACGCCCAAGTCATTTATCACTTACCGATACGAGATTCTACCATTTGCTACTTCCCGCCACGATAGGGGTTTCGATGTCATTAGACGCCTGGAAGAAGACCACAGATTCGATTACGTTTTCTACATCTTCCCATCTGAATACACAATTAGAACCATCAAACACGAGTACGTAGAAACAAAGGTGGCAGTAGTCCCGGATAGGCGCCCTTTGCGACTGCGAGCACCCGAGCTTCTCAAGGAGCAACATGGCTATTTGATCTATGAGATTGAGGGGACCGGAGGAGAATACGTTATATGTCTGAACAAGGGATCCACTGTCACACTGTTCACCACCGGGTCAGAGGCAAATAAAACATCATGGATACTCGATGCCCGGCAAATCATTGCAGGCAACGATCCAATCATAGTCTCTCAAGATAGCATCACCATGGGCAGCACAACCGTCCATCTCCGTAACCATGATTTCGATTGCCTACGGATCGTCACGAGTCGGGAGGAGCTTCTCCAAGTGGACTCGGCGTCTCGACAAACCCGCGTGCTTTCAGAAGATGCCAGTAAATGGCTCGACACCAAAGAAACAATCCATGAACATCTCCATAAGTTGGCTGAAGAACATAGGTTGCTTGGCCAGTTTGTAGTGGTGAACAATTATTCCGTGCAAGAACAAAGTGACATCGGCACGCTTTATCGTGGAGTGGGCCGTGCTCATGATCGGATGTTGTATACAGATGTGGAAGATGAAACTCTGACTCGGGATGCTGTTCTTGCCGGCGTTACAGGCGAGGATGCCTACTTCTACAATGTAAAGCAGAATGCCCTGTGGCGAGTGAATGCAGGCACACACCAAGTAATAACCAAATACTATCCGACGGTACTGGAGGACCCTGACAGCAAAATGGCAAGAGTTGAGCAGCGTGGCAACATCGTCGTGATGGCCATGAGCAGGCTCAACCACTCCGACAAGGGAGTAAATGGAATCATCTATCTCGTTCATCAAGAAAGCATGGTGCTTACTGCTGTGATCGGCAATTCAGAGATGCTCAAACGTCTTCGCCAAGCGAGTACGATTAGCCTAACAGAACTGCGAGGTGTCAATGACATGGCTCTGGTTTCCACGATGGACAAATTACCCGGCCAGAAGGTTGTCGCCACCAATGCTGAGTTTGTCACTATTCTGGCTAAGGATGAGCAAGGGAGCCAAGTCTGTTTTTGGCTGCGCACCGTGGATGGAGTTCTAATCAAGCCGAGCATCCAGCCACCCATACCAACGGACCTAACCTTGACTGTTTCATCCCACAAGGTGGCTGGACAGGATGTATTCTACTTTTACAGTATCAAGGAGCAGAGGGTCTATCGCCAACAAGGTACGGGCCAGACCAACGATACATCTGTTGGAAGCATCCCGATTCCAGGACTCTCTAATCTTTTCACCGTGTACGGTGAGGTATTCGCTACAACTAATGATGGTCGCATCCTTCGCCTGCTTGGGGACGGCTCAACATATCTTGAGGGCATCAATGAGCATTGGCTCATCAAGCATCCAAAATGGTGGCAGGACTTGGCTACCTTGGAGGCAGAGAAAGCCACGACAATTGCAGTATTCGGTGTTAGAGACCCACAAAATGCAGTGGTGCCAATTTGCAACGGTGAAGCGTGGTTGTTCGACTTGGACGGCGGCATTCTGTATAGCCAGCCGCTCGTGGATAGCCAAGCTGTGGATGCAATCTTTAGTTTGGACGGGAAGCTTATTGCACCAAACCTTATTCCACAAGTACAGCAAATGCTCAGCGGCCAGCATCTCAGAAACCCTGTCATCGTCGACGGCCGTTTGCATGTGACTACCATAGACGGTGTCGTTGTTGGATTTGACAAGAAGTCGCCAACTCTGGTTGCAGGGAACAAGGCCTGGCAGATATCTCGTCAGAACCGATTGGAAGAGGCCCTCAGAAATCTGCTGTCCAATTGGTCTGATCACAATGATGCGATTGTGTTGCAGGGTGATCCAGAATCAGCGCCGGCATGGTATTATGTTCCATCAAGAGAAATAGTGACCGCCAATGGTCTAACATGGGCGGATCATCCTCTATGGCTAGGAATGGCTGTCGGTGCTGCAACGCCATACATCTACAGCACATCTCGATGCCGTGTGTATGCTGTGGAGAGCAAAAACATGGCCAAAGACAAAGGAGAATTCGCAGCAGCACATTGCTTTGCAAAATCACTTGTGCTACAGAGCATCGTTCCCGCGGAAATAACGATTCCAATGCTGGCCGGCATCGATTTCGCACTCGTTGCAGCGACAAAAGCAACCATTGTAATAGACCGAGACAATTGGAAGCACTACAAGGCCATTGTGCTCGACAACAAGGTGGATCAGGACGGTGGCGCTAAGGTGGTAGTCGGCAGTGAGATTCCCATGAAAGTTCTGGTGCGCAAATTTCAGGGAAATCTGGAGTTCTTCGATCCCAACAATGGTAATGTTGTTATACTCCAGAATGTGTTGAAGGGAGACGGAGCTTGTTCCCAGATAGAGATCAAAGTAGGCGATGCTGAGACTATGCTCGTATCCGATTTCTCGGAGATATTCTCAAAGACGCATCCTCTGGTGGCGAGCCAAGTATTTGAACTTGGGGATGGATGTGATTGCAGTCACAAAACACAAATCGGACTATGCGGATCTGTGGGCCATTCTACGCATGACATAGCCTATATATCCCACGCATATCTTCACCGATGGCCTATATATGTCATTTCAACGTTGATAGCGTAG
SEQ ID NO.5：
ATGGAGGAGGCTGACATGGAGAACCGTGGAAACGAAGAACGGCACTCACAGTCACAGTCTGCTACACCGCTCCGCCGAAGGAAGAGATCCCCTGTCTTCGGAAGGTCTTCCGATGATATTCATTCATTCAGCAAGAATCCAGAAGAATTCTTGACTAGAAACACTCTCAGCTGCAGTTTATTAACTCGCACCGCTGAGGCCGTCACAAAAAGCCCAGCATTGCCCGCCCAAGGTTGGGTCAAAGTGGGTCGTGTGCCAGGAAGAGGAGACGTTCTCTTTGTTCAGCTGGTTGCCGAGAAATCAGATGAGACAGTGAAGGCGTACTTACTTGGCTACAATGGTGAGAACGACTTCCCCAAGCCGGCATACGTTGATATTCCAAAGAGGGGCGCCAAGAACGGAACATTTTTGTTTACCGTTGATCTCTCGGGCTGCTCCGTTGTTGTGACACAGTTGGACGAGCAGACATACCGTGTTTTCCACGACAAACGGGTGAACAGCTCCATTCTGTACGACAACGTCGTAATGTATGTGGATTTCAATCACTACCGCTATCCCAGAGCCCACGGACACGGGAGAGATACGGGACAAGCGGTCGTCTGCATGCAATTCGCCGACGGAGTGTGGAAATTGTTTCTCCAGCGGCACGAGACGGCGAATTCTCTCCCAAAGATATGGTCCCGTGGCGATGGCAAACCGGAGATTTTCAGCGACGCCGATCCCCGTCCACATATGGAAGACGGCATAAAGGAATTTCTCAATCGGCGTCGAGCTTTGCAAGACGAGTTGAAGAAATTCGCCGACATGTTTGGAGTGGACCCGGAGATAGTGTCCAATGCTGTCGACGGCAAATACGAGGGCGGCCAGTTTTCCAGGAACGATGATGCAATTCGTGGATGGAACGAATTACGTGCAAGAATGAGACAGAAAATGGCTGAAAGGAAGAAACTGTTGGAAACAAAGAAAGGTATGCTAGACTCATTCAACAACCCGGATCACCCCCTCTATGAATTTCGCAAACAAGTGCTCGACGAGCTCAAGGAAATGAGTGCATACGACAACACCTTGGAGGCATCCGAGATTGCGGACCGTACTTGGCTGTGGTTGGAGATCAAAAAAGCCAGAGGTACGAATGCGGTGGTAAACAGTGGATCAGCCCCGAAGCAGCCGAGTCTTACCGTTGGTGAACGCTACGCAGAGATGGAGGCGGCCCTTTGGATGCGTAGCGACACAGCGGCGTTTGAGCAAGGGTATGAATCTTATCAAACCGTTCGCGTGCCGGGTTACAATGAAGATATGACATCCATTGCAATGAAGAAGTTGTTCGTTGAGAGCAGTCTGACTGACGAGCAGCTGGGCGCCTTGTGCAAGCGTATATCCATCGCTAGCGATCAAGAGAACCATCTTCTGAGACAGAAGACGCAGGAGATTGAGAACACTTTCAAACAATTCTCAGACGGCAGCTTTTTGGAGTCGGCGCCTCAGGATTTCTTCCTGAGACGAGTCGGTGATAAATCTGGAGGACGCTGCTATCCTCTTGTAAGGGTAATGGCCGTCGCACTGTCTCAAGGAGAAGACGTTGCCCAGAATTTTATGGAGAACCTGAGGACATTGTCTAGGAATCCAGAGTATTCAGGCGAATCGGCAAGTTCACAAGCCACAAATGCAGGGTTGTTCAGAGATGCCCTTGCCGGCCTGCACGCGAATGTGGAAGCCATCGCCGCATCTGAGAAACTACCTGCTGTGTTTGGACTGCCCGATGTTGCTACAATGTTGAGCCAAGCGAGCGAAAGCAAGGTTTACGCTTTGAATACCGAAACACACAGCATGCTGGTTGCGGTGATTGCAGACGAACAGAGTCTGGCGCCCAAGTATTACTTCTATGATCCCAACTTTCTCATGGCCGGTTTCTCCGACATAACTCGTTTCTCAGAAGCCCTCAAGTATCACTTTGTGAATCTCCAGTTCGCTGGAAATTACAAAGCGTTTGGCACAGCGGAGGCTCCCCAGTTTAACCTGGTCCTTATTGATACGACCGCCATGGCGGATGCGCATGTTGACGGAGGGATCACCGTGAAACAATTGAGCGAGGGGGTTGATCTTACTAATGTCTTTGACAGCACCAAAAAGGCTCGTGTGGTAACTGAGGCAGCAGCTCGCTTGGCTGAAGACCAACAACTGCGCACTGGCCTCACCATAATAGAGGCTGACCAGTGGGCAAACCGGTGGCAAGATGCCGTCATACGATTGAGACTCAAGGAAGGATTAAGTGGAGAGTGGATGCCATTGCTGGAAAAAATGCAAGAAAACGCAGATGGTTCTTATCAGATTGACTTCATCAACACCAAGAATACTGGGGAAATCCGTGGGGTTTCCACTAGAGATGCGACGTTCAAAGAATTCAAATCCTACCTGGATAAAAATTTAGACGCATTGCATAAGGATTTCATCTTTCAGGATGGAGTTTTCAAACCACTAGAGGGAGAGCCCATTGACGGGTTGAATGCAATGTTCGCAGTCCAAATGATTATAGATTGGTTCAATCGGGGGACTAATGCCGAGGAAGATGCGGCCTCAGGATCCACCAATGCCCAACTTTCACTAGCGATTAAAGTGCACAGCTACATTAACATGGCCCAAGTAGCGCATACTGCAGTTACAGATGTTGCCAAGGTATACAGCCTTGTGAAGTCAGCACTGTATGGCGAGCAGGTAGCTGGGGGGGCATCGCTGTTAACATTTGGGAAGGCGCTGAAATTTGCAGCAACAGAAGGGTTGGGAGTCTTACTAGGGGCGTCCTTTGTGATCTTGGATGCCTACCAGCTGTCCCTCGTGGAAAACGATGTGCAGAAGGCCATTATTGGCACACAACTAGCCTTTGATTCCGCAAGCTTTTTAGTGAGTATCGGGGCAGCAGTTGCTGGAGTATCGGGCGTGGGCATCGTTTTGAGCGGAGCTGGCGTGTTATTGGCAGGCCTGGGAGTTGGCTTTGGAGCCTTAGCTCAGGCATTCGGGCAAATCACAGCAGATGCCCAACTGGTGGGCAAGTACTTCGCACTCTTGGACGAGGCATACAGCGGTGGCGGATACAGATACGAATCCAAAGACAAGGCATTGATGCCTCTTGCAGGAGCAGTCATCGACAAAATACGACTAGCCGACAAACATCTCCAAGTTCATTTCGACAGCCAATTCATATATCGAACCCATCATGGCAGCACAGGATCCGGCAAAATTAATTACTTCTTCTGGGCTGGCGACATGCCGCAGATGGTCCACGATCGCTCCCAAGCTATCAATGTTCGAGAGCAAATCGGGTATAAGAACGATAGTGCTACTTTGGAGCTACCGGAGGATGCCTCTGATCTGATCCTGCCTGCCACGCCCAAGTCATTTATCACTTACCGATACGAGATTCTACCATTTGCTACTTCCCGCCACGATAGGGGTTTCGATGTCATTAGACGCCTGGAAGAAGACTACAGATTCGATTACGATTTCTACATCTTCCCATCTGAATACACAATCAGAACCATCAAACACGAGTACGTAGAAACAAAGGTGGCAGTAGTCCTGGATAGGCGCTCTTTGCGACTGGGAGCACCTGAGCTTCTCAAGGAGCAACATGGCTATTTGATCTATGAGATCGAGGGGACCGGAGGACAATACGTTGTATGTCTTAACCACGGATCCACTGTCACACTGAAGACCACCGGATCAGAGGCAAATAAAACATTATGGATACTTGATGCCCGGCAACTCATTGCAGGCAACGATTCAATCACGGTTTCTCAAGATAGCATCACCATCGGCAGCACAACAGTCCATCTCCGTAACCAAGATTTCGATTGCCTACAGATCGTCATGAGTCGGGAGGAGCTTCTCCAAGTGGACTTGGCGTCTCGACAAGCCCGCGTGCTTTCAGAAGATGCCAGTAAATGGCTCGACACCAAAGAAACAATCCATGAACATCTCCATAAGTTGGCTGAAGAACATAGGTTGCTTGGCCAGTTTGTAGTGGTGAACAATTATTCCGTGCAAGAGCAAAGTGACATCGGCACGCTTTATCGTGGAGTGGGCCGTGCCTACTATGATGCTGCTCATGATCGGATGTTGTATACAGATGTGGAAGATGAAACTCTGACTCGGGATGCTGTTCTTGCCGGCGTTACAGGCGAAGATGCCTACTTCTACAATGTAAAGCAGAATGCCCTGTGGCGAGTGAATGCAGGCACACACCAAGTAATAACCAAATACTATCCGACGGTACTGGAGGACCCTGACAGCAAAATGGCAAGAGTTGAGCAGCATGGCAACATCGTCGTGATGGCCATGAGCAGGCTCAACCAGCCCGACAAGGGAGTAGATGGAATCATCTATCTCGTTCATCAAGAAAGCATGGTGCTTACTGCTGTGATCGGCAATTCAGAGATGCTCAAACGTCTTCGCCAAACGAGTACGATTAGCTTAACAGAATTGCGAGGTGTCAATGACATGGCTCTGGTTTCCACGATGGACAAATTACCCGGCCAGAAGGTTGTCGCCACCAATGCTGAGTTTGTGACTATTCTGGCTAAGGATGAGCAAGGGAGCCAAGTCTGTTTTTGGCTGCGCACCGTGGATGGAGTTCTAATCAAGCCGAGCATCCAGCCACCCATACCAACGGACCTAGCCTTGACTGTTTCATCCCACAAGGTGGCTGGACAGGATGTATTCTACTTTTGCAGTATCAAGGAGCAGACGGTCTATCGCCAACAAGGTACGGGCCAGACCAACGATACATCTGTTGGAAGCATCCCGATTCAAGGGCTCTCTAATCTTTTCACCGTGTACGGTGAGGTATTCGCTACAACTGATGATGGTCGCATCCTTCGCCTGCTTGGGGACGGCTCAACATATCTTGAGGGCATCAATGAGCATTGGCTCATCAAGCATCCAAAATGGTGGCAGGACTTGGCTACCTTGGAGGCAGAGACAGCCACGACAATTGCAGTATTCGGTGTTAGAGACCCACAAAATGCAGTGGTGCCAATTTGGTACCACAGTGGAAAGATCGCGGTGGCCTCATCAAAACTGTATGGCAAACAACTGCAATTTTTGGGGTTTCACAGCAACGATCAAGCTTGGCTGTTCGACTTGGACGGCGGCATTCTGTATAGCCAGCCGCTCGTGGATCGCCAAACTGTTGATACAATCTTTAGTTTGGACGGGAAGCTTATTGCGCCAGACCTTATTCCACAAGTACAGCAAATGCTCAGCGGCCAGCATCTCAGAAACCCTGTCATCGTGGACGGCCGTTTGCATGTGACTACCATAGACGGTGTCGTTGTTGGATTTGACAAGAAGTCGCCAACTCTGGTTGCAGTGAACAAGGCCTGGCAGATATCTCGTCAGAACCGATTGGAAGAGGCCCTCGGAAACCTGCTGTCCAATTGGTCTGATCACAATGATGCGATTGTGTTGCAGGGTGATCCAGAATCAGCGCCGGCATGGTATTATGTTCCGTCAAGAGAAATAGTGACCGCCAATGGTCTAACATGGGCGGATCATCCTCTATGGCTAGGAATGGCTGTCGGTGCTGCAACGCCATACATCTACAACACATCCCCTCGACGCCGTAGCATCGTTCCCGAGGAAATAACGATTCCAACGCTGGCTGGCATCGATTTCGCACTCGTTGCAGCGACAAATGCAACCATTGTAATAGACCGAGACAATTGGAAGCACTACAAGGCCATTGTGCTCGACAACAAGGTGGATCAGGACGGTGGCGCTAAGGTGGTACTCGGCAGTGAGATTCCCATGAAAGTTCTGGTGCGCAAATTTCAGGGAAATCTGGAGTTCTTCGATCCAAACAATGGTAATGTTGTTATACTCCAGAATGTGTTGAAGGGAGACGGAGCTTGTTGCCAGATAGACATCAAAGTAGGCGATGCTGAGACTATGCTTGCATCCGATTTCTCGGAGATATTCTCAAAGACGCATCCTCTGGTGGCAAGCCAAGTATTTGAGCTTGGGGATGTGATTGCAGTCACAAAACACAAATCGGACTATGAGGATCTGTGGGCCATTCTACACATGACATAG
the invention provides a cycadoxin optimized gene obtained by optimizing the insect-resistant cycadoxin gene, wherein the nucleotide sequence of the cycadoxin optimized gene is as follows:
SEQ ID NO.6：
atggtgatggttattgattacccttctttgagggcttctttgtatgctactaagtctgctgttccttgtaggcctcttcctggtaaccctgaaagggtttctaggagggtttctagaaggggtgctggtgaaattggtagtcatgctgatttttggatttctagagctaaggcttctgctattaggatttttcctagacaagaaggtccattgagggaaagcaggaggaggttctatttgttgagagaggctgttttcacaggtaggaggactgcatgggctgcaaatcctgctgctcagagagcagcttgggatagaaagaggtctcctttcttcggtaggtcatcagatgatattcattcattcagtaagaacccagaagagttccttactcaaaatactttgtcttgttctcttcttactagaactgctcaaactgttactaagtctcctgttttgcctgctcaaggttgggttaaggttggtagagtgccaggtagaggtgatgctcttttcgttgagcttgttgctgaaaaatctgatgaaactgttaaggcttacttgttgggttataatggtaagaatgattctcctgaacctgcttatgttgatattccaaagaggggtgcagagaatggaactttcctttttacagtggatcttagcggttgttctgttgttgttactcaattggatgagcagacttatagagtgttccatgataagagggttaattcaagtattctttacgataacgtggttatgtatgttgattttaaccattacaggtaccctagagctcatggtcatggtagagatactggtcaagctgttgtttgtatgcagtttgctgatggtgtttggaagttgttcttgcaaagacatgagactgctaattctcttccaaagatttggtctagaggtgatggtaagcctgaaattttctctgatgctgagcctagacctcatatggaggatggtattaaggagttccttaatagaaggagggctttgcaagatgaattgaagcattttgctaatactttggaagttgatcctgatattgtttctaatgctgttgatggtgaatatgaaggtggtcaattttctagaaatgatgatgcaattaggggttggaatgaattgagagcttctatgagacagaagatggctgaaagaaagaagcttcttcaaactaagaagggtatgttggatagtttcaataatccagatcatcctctttacaagttcagaaaacaagttttggatgaattgaaagagatgtcagcttatgataatacacttgaagcttctgaaatcgctgataggacttggctttggcttgaaattaagaaggctaagggaacaaatgcagttgttaatagcggagctgcacctaagcaaccatcccttactgttggtgaaagatatgctgaaatggaagcagcactttggatgagaagcgatacagctgctttcgaacaaggatatgaaagttatcaaactgttagagttccaggttataatgaagatatgacaagcattgctatgaagaagttgtttgttgaatcttccttgacagatgaacaacttggtgctctttgtaagaggatttccattgctagtgatcaagaaaatcatttgttgactcaaaagacccaagaaatcgaatcaactttcaagcaattctccgatggaagcttccttgaatctgctccacaagatttcttccttagaagagttggtgataaatccggtggtaggtgctacccacttgttagggttatggctgttgctctttctgaaggtgaagatgttgctcaaaatttcatggaaaatttgaggactttgtcaaggaatcctgaatattccggtgaatcagcttcttctcaagctacaaatgctggtcttttcagagatgctcttgctggtcttcatgcaaacgttgaagctatcgctgcatccgagaagcttcctgctgtttttggtttgcctgatgttgctactatgctttctcaggcttctgaatctaaggtttatgctcttaatactgaaactcattctatgcttgttgctgttattgttgataagcaatctttggctaggaagtattatttttacgatcctaatttccttatggctggtttctctgatattactagattctccgaagctcttaagtaccatttcgttaatttgcagtttgcaggtaattataaggctttcggtactgctgaagctcctcaattcaatcttgttcttattgatactaccgctatggaggatgctcatgttgatggtggtattactgtgaagcaactttctgaaggagttgatcttactaatgttttcgattctactaagaaggctagagtggttactgaggctgcagctaggttggctgaagatcagcaattgagaactggacttacaattattgaagctgatcagtgggctaatagatggcaggatgctgttattaggttgaagcttaaggaaggtctttctggtgagtggatgcctttgcttgaaaagatgcaagaaaatgcagatggtagttatcagattgatttcattaacactaagaacactggagaaattaggggagtttcaactagggatgctacttttaaggaattcaagtcttatttggataagaaccttgatgtgttgcataaggattttattttccaggatggagtgttcaaacctcttgagggtgagcctattgatggacttaatgctatgttcgctgttcaaatgattattgattggttcaataggggtactaacgcagaagaagatgctgcttctggttctactaacgctcaattgtctttggctattaaggttcattcttatatcaacatggcacaagtggcacatactgctgttactgatgtggcaaaggtttataatttggtgaaatctgctctttacggtgaacaagttgctggtgaaacatcattgttcactttcggtaaggctcttaaatttgctgctactgaaggtcttggtgtgttgcttggtgcttcttttgtgatccttgatgcttatcagctttctcttgttgagaatgatgttcagaaggcaattattggtactcaacttgcttttgattctgcttcttttcttgctggtattggtgctattgttgctggtgcttctggtgttggtatgattttgggtggtgctggtgttgttttgggtggtttgggtgttggttttggtgctttggctcaggcttttggtgagattactgctgatgctcagttggttggtaagtattttgctttgttggatgaggcttataggggtggtggttataggtatgagtctaaggataaggctttgatgcctttggctggtgctgtgattgataagattagacttgctgataagcatttgcaagttcattttgattctcaattcatctacaggactcatcatggttctactggttctggtaagattaattatttcttctgggctggtgatatgcctcaaatggttcatgatagatctcaagctattaatgttagggagcagattggttataagaatgataatgctactcttgaacttccagaggatgcttctgatcttattcttcctgcaactcctaagtctttcattacatatagatacgagatcttgccattcgctacttctagacatgatactggttttgatgttattagaagacttgaggaggattatagattcgattatgatttctacatcttcccttctgaatataccattagaactattaagcacgaatacgttgaaaccaaggttgctgttgtgcttgatagaagatctttgagacttggtgctcctgagcttttgaaggaacaacatggttatcttatttacgagattgaaggtacaggtggtcaatatgttatttgtttgaatcagggttctactgtgactcttaagacaactggttctgaagcaaataagactctttggattttggatgctaggcaacttattgctggtaatgatagtatcactgtttctcaagattctattactatcggttctactactgtgcatcttaggaatcaggatttcgattgtcttcagattgttatgtctagagaagaacttcttcaagttgatttggcttctggtgaagctagagttttctccgaagatgcttctaagtggcttgatacaaaggaaactattcatgaacaccttcacaagttggctgaagaacataggcttcatggtcaattcgttgttgttaataattactacaggggtgttggtagagcttattatgatgctgctcatgatagaatgctttatactgatgttgaggatgaaactcttacaagagatgctgttcttgctggtgttactggtgaagatgcttatttttataacgttaagcagaacgctttgtggagagttaacgctggtactcaccaagttattactaagtattatcctactcttcttgaggatcctgattctaacatggctagagttgagcaacatggtaatatcgttgttatggctatgtcaagattgaatcatcctgataagggtgttaacggtattatttatcttgttcatcaggaatctatcgttcttactgctgttattggaaactctgaaatgcttaagagacttagacaaacttctactatctctcttactgaacttaggggtgttaacgatatggctcttgtttcaactatggataggcttcctggtcagaaggtggtggctacaaatgcagaattcgttacacttttggctaaggatgaacaaggagctaatattaaggaacaaactgtttattgccagcaaggtactggtcaaactaatgatacttctattgaatcaatccctatccctggtttgtctaatcttcttacagtttatggtgaggttttcgctacaactaatgatggtagaatccttagattgcttggtgatggaagcacttgtcttgagggtatcaatgaacattggttgattaagcatcctaaatggtggcaggatcttgctactcttgaggcagagaaggctaccactgttgctgtttttggagttagggatttgcaaaatgctgttgttcctatttggtatcataacggtaagattgcagttgcttcatctaaactttatggtaagcaattgcagtttttgggttttaattctaacggagaagcttggcttttcgatcttgatgatggtattttgtattctcaacctttggttgattctttcctttatgctattttcagtttggatggtaagttgattgctcctatctttattcctcaggttgaacaaatgttgtccggtcaacatcttaggaaccctgttattgttgatggtaggcttcatgttactactattgatggtgttgttgttggtttcgataagaagtctcctactcttgtggctgttaataaggcttggcaaatttctagacaaaataggcttgaggaggctttgggtaatttgttgtctaattggtctgatcataacgatgctattgttcttcagggtgatcctgagtctgctcctgcttggtattatgttccttctagggagattgtgactgctaatggtttgacttgggctgatcaccctctttggcttggaatggctgttggtgctgctactccttatatttattctacttctagaaggagggtttatgttgttgagtctaagaatatggctaaggataagggtgaatttgctgctgctcattgttttgctaagtctttggttcttcagtctattgttcctgaggaaattactattccaactcttgctggtatcgatttcgctttggttgctgctactaatgctactattgttattgatagggataactggaagcattataaggcaattgttcttgataacaaggttgatcaagatggaggtgctaaggttgtggttggttctgagattcctatgaaggttcttgtgaggaagtttcaaggtaatttggaatttttcgatcctaataacggtaatgtggttattcttcaaaacgttcttaagggagatggtgcttgttgtcagattgagattaaggttggtgatgcagagactatgttggtttctgatttctctgagattttctctaagactcatcctttggttgcttctcaggttttcgaacttggtgatgttattgctgttactaagcataagagtgattatgctgatctttgggctattttgaggatgacttga
SEQ ID NO.7：
ATGGAAGAAGCTGATATGGAAAATAGAGGATACGAAGAAAGACATAGCCAAAGCCAAAGCGCTACTCCTCTTAGAAGAAGAAAGAGAAGCCCTTTCTTCGGAAGAAGCAGCGATGATATTCATAGCTTCTCAAAGAATCCTGAAGAATTCCTTACAAAAAATACTCTTTCTTGTTCTCTTCTTACTAGAACTGCTCAAACTGTTACTAAGTCTCCTGTTCTTCCTGCTCAAGGTTGGGTTAAGGTTGGTAGAGTTCCTGGTAGAGGTGATGCTCTTTCTGGTTGTTCTGTTGTTGTTACTCAACTTGATGAACAAACTTATAGAGTTTTCCATGATAAGAGAGTTAATTCTTCTATTCTTTATGATAATGTTGTTATGTATGTTGATTTCAATCATTATAGATATCCTAGAGCTCATGGTCATGGTAGAGATACTGGTCAAGCTGTTGTTTGTATGCAATTCGCTGATGGTGTTTGGAAGCTTTTCCTTCAAAGACATGAAACTGCTAATTCTCTTCCTAAGATTTGGTCTAGAGGTGATGGTAAGCCTGAAATTTTCTCTGATGCTGAACCTAGACCTCATATGGAAGATGGTATTAAGGAATTCCTTAATAGAAGAAGAGCTCTTCAAGATGAACTTAAGCATTTCGCTAATACTCTTGAAGTTGATCCTGAAATTGTTTCTAATGCTGTTGATGGTGAATATGAAGGTGGTCAATTCTCTAGAAATGATGATGCTATTAGAGGTTGGAATGAACTTAGAGCTTCTATGAGACAAAAGATGGCTGAAAGAAAGAAGAGACTTGAAAGAAATAAGGGTATGCTTGATTCTTTCAATAATCCTGATCATCCTCTTTATAAGTTCAGAAAGCAAGTTCTTGATGAACTTAAGGAAATGTCTGCTTATGATAATACTCTTGAAGCTTCTGAAATTGCTGATAGAACTTGGCTTTGGCTTGAAATTAAGAAGGCTAAGGGTACTAATGCTGTTGTTAATTCTGGTGCTGCTCCTAAGCAACCTTCTCTTACTGTTGGTGAAAGATATGCTGAAATGGAAGCTGCTCTTTGGATGAGATCTGATACTGCTGCTTTCGAACAAGGTTATGAATCTTATCAAACTCTTAGAGTTCCTGGTTATAATGAAGATATGACTTCTATTGCTATGAAGAAGCTTTTCGTTGAATCTTCTCTTACTGATGAACAACTTGGTGCTCTTTGTAAGAGAATTTCTATTGCTTCTGATCAAGAAAATCATCTTCTTACTCAAAAGACTCAAGAAATTGAATCTACTTTCAAGCAATTCTCTGATGGTTCTTTCCTTGAATCTGCTCCTCAAGATTTCTTCCTTAGAAGAGTTGGTGATAAGTCTGGTGGTAGATGTTATCCTCTTGTTAGAGTTATGGCTGTTGCTCTTTCTCAAGGTGAAGATGTTGCTCAAAATTTCATGGAAAATCTTAGAACTCTTTCTAGAAATCCTGAATATTCTGGTGAATCTGCTTCTTCTCAAGCTACTAATGCTGGTCTTTTCAGAGATGCTCTTGCTGGTCTTCATGCTAATGTTGAAGCTATTGCTGCTTCTGAAAAGCTTCCTGCTGTTTTCGGTCTTCCTGATGTTGCTACTATGCTTTCTCAAGCTTCTGAATCTAAGGTTTATGCTCTTAATACTGAAACTCATTCTATGCTTGTTGCTGTTATTGTTGATAAGCAATCTCTTGCTAGAAAGTATTATTTCTATGATCCTAATTTCCTTATTGCTGGTTTCTCTGATATTACTAGATTCTCTGAAGCTCTTAAGTATCATTTCGTTAATCTTCAATTCGCTGGTAATTATAAGGCTTTCGGTACTGCTGAAGCTCCTCAATTCAATCTTGTTCTTATTGATACTACTGCTATGGCTGATGCTCATGTTGATGGTGGTATTACTGTTAAGCAACTTTCTGAAGGTGTTGATCTTACTAATGTTTTCGATTCTACTAAGAAGGCTAGAGTTGTTACTGAAACTGCTGCTAGACTTGCTGAAGATCAACAACTTAGAACTGGTCTTACTATTATTGAAGCTGATCAATGGGCTAATAGATGGCAAGATGCTGTTATTAGACTTAAGCTTAAGGAAGGTCTTTCTGGTGAATGGATGCCTCTTCTTGAAAAGATGCAAGAAAATGCTGATGGTTCTTATCAAATTGATTTCATTAATACTAAGAATACTGGTGAAATTAGAGGTGTTTCTACTAGAGATGCTACTTTCAAGGAATTCAAGTCTTATCTTGATAAGAATCTTGATGTTCTTCATAAGGATTTCATTTTCCAAGATGGTGTTTTCAAGCCTCTTGAAGGTGAACCTATTGATGGTCTTAATGCTATGTTCGCTGTTCAAATGATTATTGATTGGTTCAATAGAGGTACTAATGCTGAAGAAGATGCTGCTTCTGGTTCTACTAATGCTCAACTTTCTCTTGCTATTAAGGTTCATTCTTATATTAATATGGCTCAAGTTGCTCATACTGCTGTTACTGATGTTGCTAAGGTTTATAATCTTGTTAAGTCTGCTCTTTATGGTGAACAAGTTGCTGGTGAAGCTTCTCTTCTTACTTTCGGTAAGGCTCTTAAGTTCGCTGCTACTGAAGGTCTTGGTGTTCTTCTTGGTGCTTCTTTCGTTATTCTTGATGCTTATCAACTTTCTCTTGTTGAAAATGATGTTCAAAAGGCTATTATTGGTACTCAACTTGCTTTCGATTCTGCTTCTTTCCTTGCTGGTATTGGTGCTATTGTTGCTGGTGCTTCTGGTGTTGGTATGATTCTTGGTGGTGCTGGTGTTGTTCTTGGTGGTCTTGGTGTTGGTTTCGGTGCTCTTGCTCAAGCTTTCGGTGAAATTACTGCTGATGCTCAACTTGTTGGTAAGTATTTCGCTCTTCTTGATGAAGCTTATAGAGGTGGTGGTTATAGATATGAATCTAAGGATAAGGCTCTTATGCCTCTTGCTGGTGCTGTTATTGATAAGATTAGACTTGCTGATAAGCATCTTCAAGTTCATTTCGATTCTCAATTCATTTATAGAACTCATCATGGTCTTACTGGTTCTGGTAAGATTAATTATTTCTTCTGGGCTGGTGATATGCCTCAAATGGTTCATGATAGATCTCAAGCTATTAATATTAGAGAACAAATTGGTTATAAGAATGATCATGCTACTCTTGAACTTCCTGAAGATGCTTCTGATCTTATTCTTCCTGCTACTCCTAAGTCTTTCATTACTTATCAATATCAAATTCTTCCTTTCGCTACTTCTAGACATGATAGAGGTTTCGATGTTATTAGAAGACTTGAAGAAGATTATAGATTCGATTATGATTTCTATATTTTCCCTTCTGAATATACTATTAGAACTATTAAGCATGAATATGTTGAAACTAAGGTTGCTGTTGTTCTTGATAGAAGATCTCTTAGACTTGGTGCTCCTGAACTTCTTAAGGAACAACATGGTTATCTTATTTATGAAATTGAAGGTACTGGTGGTCAATATGTTATTTGTCTTAATCAAGGTTCTACTGTTACTCTTAAGACTACTGGTTCTGAAGCTAATAAGACTCTTTGGATTCTTGATGCTAGACAACTTATTGCTGGTAATGATTCTATTACTGTTTCTCAAGATTCTATTACTATTGGTTCTACTACTGTTCATCTTAGAAATCAAGATTTCGATTGTCTTCAAATTGTTATGTCTAGAGAAGAACTTCTTCAAGTTGATCTTGCTTCTGGTGAAGCTAGAGTTTTCTCTGAAGATGCTTCTAAGTGGCTTGATACTAAGGAAACTATTCATGAACATCTTCATAAGCTTGCTGAAGAACATAGACTTCATGGTCAATTCGTTGTTGTTAATAATTATTATAGAGGTGTTGGTAGAGCTTATTATGATGCTGCTCATGATAGAATGCTTTATACTGATGTTGAAGATGAAACTCTTACTAGAGATGCTGTTCTTGCTGGTGTTACTGGTGAAGATGCTTATTTCTATAATGTTAAGCAAAATGCTCTTTGGAGAGTTAATGCTGGTACTCATCAAGTTATTACTAAGTATTATCCTACTCTTCTTGAAGATCCTGATTCTAAGATGGCTAGAGTTGAACAACATGGTAATATTGTTGTTATGGCTATGTCTAGACTTAATCATCCTGATAAGGGTGTTAATGGTATTATTTATCTTGTTCATCAAGAATCTATGGTTCTTACTGCTGTTATTGGTAATTCTGAAATGCTTAAGAGACTTAGACAAACTTCTACTATTTCTCTTACTGAACTTAGAGGTGTTAATGATATGGCTCTTGTTTCTACTATGGATAAGCTTCCTGGTCAAAAGGTTGTTGCTACTAATGCTGAATTCGTTACTATTCTTGCTAAGGATGAACAAGGTTCTCAAGTTTGTTTCTGGCTTAGAACTGTTGATGGTGTTCTTATTAAGCCTTCTATTCAACCTCCTATTCCTACTGATCTTACTCTTACTGTTTCTTCTCATAAGGTTGCTGGTCAAGATGTTTTCTATTTCTATTCTATTAAGGAACAAACTGTTTATTGTCAACAAGGTACTGGTCAAACTAATGATACTTCTATTGAATCTATTCCTATTCCTGGTCTTTCTAATCTTCTTACTGTTTATGGTGAAGTTTTCGCTACTACTGATGATGGTAGAATTCTTAGACTTCTTGGTGATGGTTCTACTTATCTTGATGGTATTAATGAACATTGGCTTATTAAGCATCCTAAGTGGTGGCAAGATCTTGCTACTCTTGAAGCTGAAAAGGCTACTACTGTTGCTGTTTTCGGTGTTAGAGATCTTCAAAATGCTGTTGTTCCTATTTGGTATCATAATGGTAAGATTGCTGTTGCTTCTTCTAAGCTTTATGGTAAGCAACTTCAATTCCTTGGTTTCAATTCTAATGGTGAAGCTTGGCTTTTCGATCTTGATGATGGTATTCTTTATTCTCAACCTCTTGTTGATTCTTTCCTTTATGCTATTTTCTCTCTTGATGGTAAGCTTATTGCTCCTATTTTCATTCCTCAAGTTGAACAAATGCTTTCTGGTCAACATCTTAGAAATCCTGTTATTGTTGATGGTAGACTTCATGTTACTACTATTGATGGTGTTGTTGTTGGTTTCGATAAGAAGTCTCCTACTCTTGTTGCTGTTAATAAGGCTTGGCAAATTTCTAGACAAAATAGACTTGAAGAAGCTCTTGGTAATCTTCTTTCTAATTGGTCTGATCATAATGATGCTATTGTTCTTCAAGGTGATCCTGAATCTGCTCCTGCTTGGTATTATGTTCCTTCTAGAGAAATTGTTACTGCTAATGGTCTTACTTGGGCTGATCATCCTCTTTGGCTTGGTATGGCTGTTGGTGCTGCTACTCCTTATATTTATTCTACTTCTAGAAGAAGAGTTTATGCTGTTGAATCTAAGCATATGGCTAAGGATAAGGGTGAATTCGCTGCTGCTCATTGTTTCGCTAAGTCTCTTGTTCTTCAATCTATTGTTCCTGAAGAAATTACTATTCCTACTCTTGCTGGTATTGATTTCGCTCTTGTTGCTGCTACTAATGCTACTATTGTTATTCATAGAGATAATTGGAAGCATTATAAGGCTATTGTTCTTGATAATAAGGTTGATCAAGATGGTGGTGCTAAGGTTGTTGTTGGTTCTGGTATTCCTATGAAGGTTCTTGTTAGAAAGTTCCAAGGTAATCTTGAATTCTTCGATCCTAATAATGGTAATGTTGTTATTCTTAGAAATGTTCTTAAGGGTGATGGTGCTTGTTCTCAAATTGAAATTAAGGTTGGTGATGCTGAAACTATGCTTGTTTCTGATTTCTCTGAAATTTTCTCTAAGACTCATCCTCTTGTTGCTTCTCAAGTTTTCGAACTTGGTGATGTTATTGCTGTTACTAAGCATAAGTCTGATTATGCTGATCTTTGGGCTATTCTTAGAATGACTTGA
SEQ ID NO.8：
ATGGCAGTTGCTTTGAGCCAAGGTGAAGATGTTGCTCAGAATTTCATGGAGAATTTGAGGACTTTGAGCAGGAATCCTGAATATAGCGGTGAAAGCGCTTCCTCTCAGGCTACCAATGCTGGTCTTTTTAGGGATGCTTTGGCTGGTCTTCATGCTAACGTTGAAGCTATCGCTGCTTCTGAGAAGTTGCCTGCTGTGTTCGGATTGCCTGATGTTGCTACTATGCTTTCTCAAGCTTCTGAGTCTAAGGTTTATGCTCTTAATACTGAAACTCATTCTATGCTTGCTGCTGTTATCGTTGATAAGCAATCTTTGGCTAGGAAGTATTATTTCTATGATCCTAATTTCCTTATGGCTGGTTTCTCTGATATTACTAGATTCTCCGAAGCTCTTAAGTATCATTTTGTTAATCTTCAGTTCGCTGGTAATTATAAAGCTTTCGGTACTGCTGAAGCTCCTCAATTCAATCTTGTTCTTATTGATACTACCGCTATGGCTGATGCTCATGTTGATGGTGGTATTACTGTGAAACAACTTTCTGAGGGTGTGGATTTGACTAATGTTTTTGATTCTACTAAGAAGGCTAGAGTGGTTACCGAGGCAGCAGCTAGGTTGGCTGAAGATCAGCAATTGAGAACTGGATTGACTATTATTGAAGCAGATCAATGGGCTAATAGATGGCAGGATGCTGTGATTAGATTGAAGCTTAAGGAAGGACTTTCTGGTGAGTGGATGCCTCTTCTTGAAAGAATGCAAGAAAATGGACACGGTAGCTATCAAATTGAATTTCTTAATACCAAGAACACCGGTGAAACAAGATGGGTTAGCACAAGAGATGCTACTTTTAAGGAATTCAAGTCTTATCTTGATAAGAACTTCGATGCTTTGAGGAAGGATTTCATTTTCCAAGATGGAGTTTTCAAACCTCTTGATGGTGAGCCTATTGATGGTCTTAATGCTATGTTCGCTGTTCAAATGATTATTGATTGGTTCAATCCTGGTACTAATGCTGAGGAGGATGCTGCTAGTGGTTCTACTAATGCTCAACTTTCTTTGGCTATTAAGGTGCATTCTTATATTAACATGGCTCAAGTTGCTCACACTGCTGTTACTGATGTTGCAAAGGTTTATAATCTTGTTAAGTCTGCTCTTTACGGTGAACAAGTTGCTGGTGAAGCTAGCTTGCTTACTTTCGGTAAGGCTCTTAAGTTCGCTGCTACTGAAGGTTTGGGTGTTCTTTTGGGTGCTTCTTTCGTTATTCTTGATGCTTATCAGCTTTCTCTTGTTGAGAATGATGTTCAGAAGGCAATTATTGGAACTCAATTGGCTTTTGATTCTGCTTCTTTTTTGGCTGGTATTGGTGCTATTGTTGCTGGTGCTTCTGGTGTTGGTATGATTTTGGGTGGTGCTGGTGTTATTTTGGGTGGATTGGGTGTTGGTTTTGGTGCTTTGGCTCAGGCTTTTGGTCAGATTACTGCTGATGCTCAGTTGGTTGGTGAGTATTTTGCTTTGTTGGATGAGGCTTATTCTGGTGGTGGTTATAGGTATGAGTCTAAGGATAAGGCTTTGATGCCTCTTGCTGGTGCTGTGATTGATAAGATTAGGCTTGCTGATAAGAGGTTGCAAGTTCATTTTGATTCTCAATTTATCTACAGGACTCATCACGGTTCTACTGGTTCTGGTAAGATTAATTATTTCTTCTGGGTTGGTGATATGCCTCAAATGGTTCATGATAGATCTCAAGCTATTAATGTTAGGGAACAGATTGGTTATAAGAATGATTCTGCTACTTTGGAATTGCCTGAAGATGCTTCTGATTTGATTTTGCCTGCTACTCCTAAGTCTTTCATTACATATAGATACGAAATCCTTCCATTCGCTACATCTAGACATGATAGAGGTTTTGATGTTATTAGAAGATTGGAAGAAGATTACAGGTTCGATTATGATTTCTACATTTTCCCTTCCGAGTACACTATTAGAACTATTAAGCATGAATACGTGGAAACTAAGGTTGCTGTTGTTCTTGATAGAAGATCTTTGAGACTTGGTGCTCCTGAACTTTTGAAGGAGCAGCATGGTTATTTGATTTACGAGATTGAAGGAACTGGTGGTCAATATGTTATTTGTTTGAATCATGGTTCCACTGTTACTCTTAAGACTACTGGTTCCGAAGCTAATAAGACACTTTGGATTTTGGATGCTAGGCAGCTTATTGCTGGAAATGATTCTATCACTGTTTCTCAAGATTCTATTACTATCGGTTCTACTACTGTTCACCTTAGAAATCAAGATTTCGATTGCCTTCAAATTGTGATGTCTAGGGATGAACTTCTTCAAGTGGATCTTGCTTCTAGGGAAGTTAGGGTTTTCTCCGAAGATGCTTCAAAATGGCTTGATACCAAGGAAACTATTCATGAACATCTTCACAAGTTGGCTGGTGAGCATAGACTTCATGGTCAATTCGTTGTTGTTAATAATTACTACAGGGGTGTGGGTAGAGCTTACTATGATGCTGCACATGATAGAATGCTTTATACTGATGTTGAAGATGAAACTCTTACAAGAGATGCAGTTCTTGCTGGTGTTACTGGAGAAGATGCTTATTTTTATAACGTTAACCAGAACGCTCTTTGGAGGGTTAACGCTGATACTCATCAAGTTATTACTAAGTATTACCCTACCTTGTTGGAGGATCCTGATTCTAAAATGGCTAGAGTTGAGCAACATGGAAATATCGTTGTTATGGCTATGTCAAGATTGAATCATCCTGATAAGGGTGTTAACGGTATTATTTATCTTGTTCATCAGGAATCTATGGTTCTTACTGCTGTTATTGGTAATTCTGAAATGCTTAAGAGGTTGAGACAAACTTCTACTATCTCTCTTACTGAACTTAGGGGTGTTAACGATATGGCTCTTGTTTCTACTATGGATAGACTTCCTGGTCAAAAGGTGGTTGCTACTAATGCTGAATTCGTGACACTTCTTGCTAAGGATGAACAAGGTTCTCAAGTGTGTTTTTGGTTGAGGACTGTTGATGGTGTTTTGATTAAGCCTTCTATTCAGCCTCCTATTCCTACTGATCTTACTCTTACTGTTTCTTCTCATAAGGTTGCTGGTCAGGATGTTTTTTATTTTTATTCTATCAAGGAGCAGACCGTTTATTGTCAACAAGGTACCGGACAAACCAACGATACCTCTATTGAATCTATTCCTATTCCTGGTTTGTCTAATCTTCTTACTGTTTATGGTGAGGTTTTTGCTACTACTAATGATGGTAGAATTCTTAGACTTTTGGGTGATGGAAGCACTTATCTTGAAGGTATTAATGAGCATTGGTTGATTAAGCATCCTAAGTGGTGGCAGGATTTGGCTACTTTGGAGGCAGAGAAGGCTACAACCGTTGCAGTTTTTGGTGTGAGGGATCCTCAAAATGCTGTGGTGCCTATTTGGTATCATAACGGAAAAATTGCTGTTGCTTCAAGCAAGCTTTATGGTAAGCAATTGCAATTCCTTGGATTTAATTCTAACGGTGAAGCATGGCTTTTCGATCTTGATGATGGTATTTTGTATTCTCAACCTTTGGTTGATTCTCAAGCTGTTGATGCTATTTTTTCTCTTGATGGTAAGCTTATTGCTCCAAATTTGATTCCTCAAGTTGAACAGATGTTGTCTGGTCAACACCTTAGGAATCCTGTTATTGTGGATGGTAGGTTGCATGTTACTACTATTGATGGTGTTGTTGTTGGTTTCGATAAGAAGTCTCCTACTCTTGTGGCTGTTAATAAGGCTTGGCAAATTTCTAGACAAAATAGGTTGGAAGAGGCTTTGGGTAATTTGTTGTCTAATTGGTCTGATCATAACGATGCTATTGTTCTTCAGGGTGATCCTGAGTCTGCTCCTGCTTGGTATTATGTTCCTTCTAGGGAGATTGTGACTGCTAATGGTTTGACTTGGGCTGATCACCCTCTTTGGCTTGGAATGGCTGTTGGTGCTGCTACTTTGTATATTTATTCTACTTCTAGGAGGAGAGTTTATGCTGTTGAGTCTAAGAAGATGGCTAAGGATAAAGGTGAATTTGCTGCTGCTCATTGTTTTGCTAAGTCTCTTGTTCTTCAATCTATTGTTCCTGAAGAAATTACAATCCCAACTTTGGCTGGTACTGATTTCGCTTTGGTTGCTGCTACTAATGCAACTATTGTTATTGATAGAGATAACTGGAAGCATTATAAGGCTATTGTGTTGGATAATAAGGTTGATCAAGATGGAGGTGCTAAGGTTGTGGTTGGTTCTGAGATTCCTATGAAGGTTTTGGTTAGAAAGTTTCAAGGTAACTTGGAATTCTTCGATCCTAATAATGGTAATGTTGTTATCCTTCAAAACGTTCTTAAGGGAGATGGTGCTTGTTCTAAGATTGAAATTAAAGTTGGTGATGCTGAAACTATGCTTGTTAGTGATTTCTCTGAGATTTTCTCTAAGACTCATCCTTTGGTTGCTTCTCAGGTTTTCGAACTTGGTGATGTTATTGCAGTTACTAAGCATAAGTCTGATTATGCTGATCTTTGGGCTATTTTGAGGATGACTTGA
SEQ ID NO.9：
ATGGAAGAAGTTGATATGCAAAACAGAGGATACGAAGAATGGCACAGCCAAAGCCAAAGCGCTACTACTTTGAGAAGAAGAAAGAGAAGCCCTGTGTTCGGTAGGAGCAGCGATGATATTCATAGCTTCAGCAAGAATCCAGAAGAATTCCTTACTCAAAACACACTTTCATGTTCACTTCTTACTAGGACAGCTGAGACAGTTACCAAGTCTCCTGTTCTTCCAGAAAGAGGATGGGTTAAAGTGGGTAGAGTTCCAGGTAGAGGTGATGCTCTTTTCGTTCAAGTTGTTGCTGAACAATCATATGATACTGTGAAGGCTTATTTGCTTGGTTATAATGGTGAGAATGATTCTCCTAATCCTGCTTATGTTGATATTCCTAAGAGGGCTGCTGAGAATGGTACTTTCCTTTTTACTGTTGATTTGTCTGGTTGTTCTGTTGTTGTTACTCAGTTGGATGAGCAAACTTATAGAGTGTTTCATGATAAGAGAGTTAACTCATCAATCCTTTATGATAACGTTGTTATGTACGTTGATTTCAATCATTACAGGTATCCTAGGGCTCACGGTCATGGTAGAGATACTGGTCAAGCTGTTGTTTGTATGCAATTTGCTGATGGTGTTTGGAAGCTTTTCCTTCAAAGACATGAGACTGCTAATTCTCTTCCTAAGATTTGGTCTAGAGGTGATGGTAAACCTGAAATTTTCTCTGATGCTGAGCCTAGACAACATATGGAGGATGGTATCAAGGAGTTCCTTAATAGAAGGAGAGCTTTGCAAGATGAATTGAAGAAGTTCGCAGATATGTTCGGAGTTGATCCTGAGATTGTTTCTAATGCTGTGGATGGTAAGTATAAGGGTGGTCAATTTTCTAGAAATGATGATGCAATTAGGGGTTGGAATGAATTGAGAGCTTCAATGAGGCAAAAGATGGCTGAAAGAAACAAGGTGTTGCTTACTATGAAGTTGGAACAACCTAGAAGCAGCAGGGTTTTGCCTTTGGTGAATGCTACTCAAAGATGGAGGAGGCCTTTCGGTTGTGTTGCAACTCAAAGAAGACTTTCTAAGGCTGGTAGATTGGTTCAGGCTTATATTCATAGGGATCAGGAGAATCATTTGTTGACTCAAAAGACTCAAGAGATTGAGTCCACTTTCAAGCAATTCTCAGATGGTTCATTTCTTGAATCTGCTCCACAAGATTTCTTCCTTAGGAGAGTGGGTGATAAGTCTGGTGGTAGGTGTTACCCACTTGTTAGAGTTATGGCTGTTGCTCTTTCTCAGGGTGAAGATGTTGCTCAAAATTTCATGGAAAACCTTAGGACTCTTTCAAAGAATCCTGAGTATTCTGGTGAATCTGCTTCTTCTCAAGCTACAAATGCTGGTCTTTTCAGAGATGCTCTTGCTGGTCTTCATGCTAACGTTGAAGCTATTGCTGCTTCTGAAAAGTTGCCTGCTGTGTTCGGTTTGCCTGATGTTGCTACTATGCTTTCCCAAGCTTCTGAATCTAAGGTTTATGCTCTTAATACTGAAACTCATTCTATGCTTGCTGCTGTTATTGTTGATAAGCAATCTTTGGCTAGGAAGTATTATTTTTACGATCCTAATTTCCTTATGGCTGGTTTCTCTGATATTACTAGATTCTCCGAAGCTCTTAAGTACCATTTTGTTAATCTTCAGTTCGCAGGTAATTATAAGCCATTCGGTACTGCTGAAGCTCCTCAATTCAATCTTGTTCTTATTGATACTACCGTTATGGCTGATGCACATGTTGATGGTGGTATTACTGTTAAGCAACTTTCTGAGGGTGTGGATCTTACTAATGTTTTCGATTCTACTAAGAAGGCTAGAGTTGTTACTGAAGCAGCTGCTAGGTTGGCTGAAGATCAGCAATTGAGAACTGGACTTACCATTATTGAAGCTGATCAATGGGCTAATAGATGGCAGGATGCTGTTATTAGACTTAAGCTTAAGGAAGGACTTTCTGGTGAGTGGATGCCTTTGCTTGAAAAGATGCAAGAAAATGCAGATGGTAGTTATCAAATCGATTTCATCAACACTAAGAACACTGGTGAGATTAGAGGTGTTTCAACTAGAGATGGAACTTTCAAGGAACTTAAGTCTTATTTGGATAAGAACTTGGATGCTTTGCACAAAGATTTCATTTTTCAAGATGGTGTTTTCAAGCCTTTGGAGGGTGAGCCTATTGATGGTCTTAATGCAATGTTCGCTGTTCAAATGATTATTGATTGGTTCAATAGGGGTACTAATGCTGAAGAGGATGCTGCTTCTGGTAGTACTAACGCTCAGTTGTCTTTGGCTATTAAGGTTCATTCTTATATCAACATGGCTCAAGTTGCTCATACTGCTGTTACTGATGTTGCTAAGGTTTATTCACTTGTTAAGTCTGCTTTGTACGGTGAACAAGTTGCTGGTGGTGCTTCATTGCTTACTTTCGGTAAGGCTTTGAAGTTGGCTGCTACTGAAGGTCTTGGTGTTCTTTTGGGTGCTCCTGTTGTTATTCTTGATGCTTATCAGCTTTCTCTTGTTGAAAATGATGTTCAGAAGGCTATTATTGGTACTCAGTTGGATTTCGATTCTGCTTCTTTTCTTGTTTCTATTGGTGCTGCTGTTGCTGGTGTTTCTGGTGTTGGTATTGTTCTTTCTGGTGCTGGTGTTTTGTTGGCTGGTTTGGGTGTTGGTTTTGGTGCTTTGGCTCAGGCTTTTGGTGAGATTACTGCTGATGCTCAGTTGGTTGGTAAGTATTTTGCTTTGTTGGATGAGGCTTATAGGGGTGGTGGTTATAGGTATGAGTCTAAGGATAAGGCTTTGATGCCTTTGGCTGGTGCTGTGATTGATAAGATTAGACTTGCTGATAAGCATTTGCAAGTTCATTTTGATTCTCAATTCATCTACAGGACTCATCATGGTTCTACTGGTTCTGGTAAGATTAATTATTTCTTCTGGGTTGGTGATATGCCTAAGATGGTTCATGATAGATCTCAAGCTATTAATGTTAGGGAACAGATTGGTTATAAGAATGATTCTGCTACTTTGGAATTGCCTGAAGATGCTTCTGATTTGATTTTGCCTGCTACTCCTAAGTCTTTCATTACATATAGATACGAAATCCTTCCATTCGCTACATCTAGACATGATAGAGGTTTTGATGTTATTAGAAGATTGGAAGAAGATCATAGATTCGATTATGTTTTCTACATCTTCCCTTCCGAATATACAATTAGAACTATTAAGCACGAGTACGTTGAAACTAAGGTGGCTGTGGTTCCTGATAGAAGACCTCTTAGGTTGAGAGCACCTGAACTTCTTAAGGAACAACATGGTTATCTTATTTACGAGATTGAAGGAACAGGTGGTGAATATGTTATTTGTTTGAATAAGGGTTCCACTGTGACACTTTTTACTACTGGTTCTGAAGCTAATAAGACTTCTTGGATCCTTGATGCAAGGCAAATTATTGCTGGTAATGATCCTATTATCGTTTCTCAGGATTCTATTACTATGGGTTCTACTACCGTTCACCTTAGGAATCATGATTTCGATTGCCTTAGAATTGTTACTTCTAGAGAGGAGCTTCTTCAAGTTGATTCTGCTTCAAGACAAACTAGGGTTCTTTCTGAAGATGCTTCAAAGTGGCTTGATACTAAAGAAACTATTCATGAGCATCTTCATAAGCTTGCTGAAGAGCATAGGCTTCTTGGTCAATTCGTTGTTGTGAATAACTATTCTGTTCAAGAACAGTCTGATATTGGAACTCTTTACAGAGGTGTTGGTAGAGCACATGATAGAATGCTTTATACTGATGTTGAGGATGAAACTCTTACTAGGGATGCTGTGCTTGCTGGTGTTACTGGTGAAGATGCTTACTTTTATAATGTTAAGCAGAACGCTTTGTGGAGAGTTAATGCTGGTACTCACCAAGTTATTACTAAGTATTATCCTACAGTGCTTGAAGATCCTGATTCTAAAATGGCTAGAGTTGAGCAAAGAGGTAATATCGTTGTTATGGCTATGTCAAGACTTAATCATTCTGATAAGGGTGTTAATGGTATTATTTACCTTGTTCACCAAGAATCTATGGTTCTTACTGCTGTTATTGGAAATTCAGAAATGCTTAAGAGGCTTAGACAAGCTAGCACAATCTCCCTTACTGAACTTAGGGGAGTGAACGATATGGCTTTGGTTTCTACTATGGATAAGCTTCCTGGTCAGAAGGTTGTTGCTACTAATGCTGAGTTCGTTACAATTCTTGCAAAGGATGAACAGGGATCTCAAGTTTGTTTTTGGCTTAGAACTGTTGATGGTGTTCTTATTAAGCCTTCTATTCAACCACCTATCCCTACAGATCTTACTCTTACTGTTTCTTCTCATAAAGTTGCTGGTCAAGATGTTTTCTATTTTTATTCCATCAAGGAGCAAAGGGTTTATAGACAACAAGGTACCGGACAAACAAATGATACCTCTGTTGGATCTATTCCTATCCCTGGACTTTCTAATCTTTTCACTGTTTATGGTGAGGTTTTTGCTACAACTAATGATGGTAGAATTCTTAGACTTCTTGGTGATGGATCTACTTATCTTGAGGGTATTAACGAACATTGGCTTATTAAGCATCCTAAGTGGTGGCAAGATTTGGCTACTCTTGAAGCAGAGAAGGCTACAACCATTGCTGTTTTCGGTGTTAGGGATCCTCAAAATGCTGTGGTGCCTATTTGTAATGGTGAGGCTTGGCTTTTCGATCTTGATGGTGGTATCCTTTATTCTCAACCTTTGGTTGATTCTCAGGCTGTTGATGCTATTTTCTCTCTTGATGGTAAGCTTATTGCTCCTAATCTTATTCCTCAAGTTCAACAAATGCTTTCTGGTCAGCATCTTAGGAATCCTGTTATTGTTGATGGTAGGCTTCATGTTACTACCATTGATGGTGTTGTTGTTGGTTTCGATAAGAAGTCTCCTACTCTTGTTGCTGGTAATAAGGCTTGGCAAATTTCTAGACAAAATAGGCTTGAGGAAGCTTTGAGAAATTTGTTGTCTAATTGGTCTGATCATAACGATGCTATTGTTCTTCAAGGTGATCCTGAGTCTGCTCCAGCATGGTATTATGTTCCTTCAAGAGAGATTGTGACTGCTAATGGTTTGACTTGGGCTGATCACCCTCTTTGGCTTGGTATGGCTGTTGGTGCTGCAACTCCTTATATTTATTCTACTTCTAGATGCAGAGTGTATGCTGTGGAATCTAAGAATATGGCTAAGGATAAAGGTGAATTTGCTGCTGCTCATTGTTTCGCTAAGTCTTTGGTTCTTCAATCTATTGTTCCTGCTGAAATTACTATTCCTATGCTTGCTGGTATTGATTTCGCTTTGGTTGCTGCTACTAAGGCTACTATTGTTATTGATAGAGATAACTGGAAGCATTATAAGGCTATTGTTTTGGATAACAAGGTGGATCAAGATGGTGGTGCTAAGGTTGTTGTTGGAAGTGAGATTCCTATGAAGGTTTTGGTGAGAAAGTTTCAAGGTAATTTGGAATTTTTCGATCCTAATAACGGTAATGTTGTTATTTTGCAAAACGTTCTTAAGGGTGATGGTGCTTGTTCTCAGATTGAAATTAAAGTTGGTGATGCTGAGACTATGCTTGTGTCTGATTTCTCTGAAATTTTTTCTAAGACCCATCCTCTTGTTGCTTCTCAGGTTTTCGAATTGGGTGATGGTTGTGATTGTAGTCATAAGACTCAAATTGGTTTGTGTGGTTCTGTTGGTCATTCTACTCATGATATTGCTTATATTTCCCATGCTTATTTGCATAGATGGCCTATTTATGTTATCTCTACTTTGATTGCATGA
SEQ ID NO.10：
ATGGAAGAAGCTGATATGGAAAATAGAGGAAATGAAGAAAGACATAGCCAAAGCCAAAGCGCTACTCCTTTGAGAAGAAGAAAGAGAAGCCCTGTTTTCGGTAGAAGCAGCGATGATATTCATAGCTTCAGCAAGAATCCTGAAGAATTCCTTACTAGAAACACACTTTCATGTTCACTTTTGACCAGGACAGCTGAAGCAGTTACAAAGTCTCCAGCTCTTCCTGCACAAGGATGGGTTAAAGTTGGTAGAGTTCCTGGAAGAGGTGATGTTCTTTTCGTTCAACTTGTTGCTGAAAAGTCTGATGAAACTGTTAAAGCTTATTTGCTTGGTTATAACGGTGAGAATGATTTTCCTAAGCCTGCTTATGTTGATATTCCTAAGAGGGGTGCTAAGAATGGTACTTTTCTTTTTACAGTGGATCTTTCTGGTTGTTCTGTTGTTGTTACTCAATTGGATGAGCAAACTTATAGAGTGTTCCATGATAAGAGGGTTAATTCTTCAATTCTTTACGATAACGTGGTTATGTATGTTGATTTTAACCATTACAGGTACCCTAGGGCTCACGGTCATGGTAGAGATACTGGTCAAGCTGTTGTTTGTATGCAATTTGCTGATGGTGTTTGGAAGTTGTTCTTGCAAAGACATGAGACTGCTAATTCTCTTCCTAAGATTTGGTCTAGAGGTGATGGTAAACCTGAAATTTTCTCTGATGCTGATCCTAGACCTCATATGGAGGATGGTATTAAGGAGTTCCTTAATAGAAGGAGGGCTTTGCAAGATGAATTGAAGAAGTTCGCAGATATGTTCGGAGTTGATCCTGAAATTGTTTCTAATGCAGTGGATGGTAAGTATGAAGGTGGACAATTTTCTAGAAATGATGATGCTATTAGGGGTTGGAACGAATTGAGAGCTAGAATGAGACAGAAGATGGCTGAAAGGAAGAAGCTTCTTGAAACTAAGAAGGGAATGTTGGATAGCTTCAATAATCCAGATCATCCTCTTTATGAATTCAGGAAGCAAGTTTTGGATGAATTGAAAGAAATGAGCGCTTATGATAATACTCTTGAAGCTAGCGAGATTGCTGATAGGACTTGGTTGTGGCTTGAAATTAAGAAGGCTAGAGGAACTAACGCAGTTGTTAATAGCGGAAGCGCACCTAAGCAACCTTCCCTTACTGTTGGTGAAAGATATGCAGAGATGGAAGCAGCACTTTGGATGAGAAGCGATACAGCTGCTTTCGAACAAGGTTATGAAAGTTACCAAACTGTTAGAGTTCCTGGTTATAATGAAGATATGACTAGCATTGCTATGAAGAAGCTTTTTGTTGAAAGTAGCCTTACAGATGAACAACTTGGTGCTCTTTGTAAGAGGATTTCTATTGCTAGTGATCAAGAAAATCACCTTTTGAGACAGAAGACTCAAGAAATTGAGAATACCTTCAAGCAATTCTCCGATGGAAGCTTCCTTGAAAGTGCTCCACAAGATTTCTTCCTTAGGAGAGTTGGTGATAAGAGCGGTGGTAGGTGTTATCCTTTGGTTAGAGTTATGGCTGTTGCTCTTTCTCAAGGTGAAGATGTTGCTCAAAATTTCATGGAAAATTTGAGGACTCTTAGTAGGAATCCTGAGTATTCTGGTGAGTCAGCTTCTTCTCAGGCTACCAATGCTGGTCTTTTTAGAGATGCTCTTGCTGGTTTGCATGCTAACGTTGAAGCTATCGCTGCTTCTGAAAAGCTTCCAGCTGTTTTCGGTTTGCCTGATGTTGCTACTATGCTTTCTCAAGCTTCTGAATCTAAGGTTTATGCTCTTAATACTGAAACTCACTCTATGCTTGTTGCTGTTATTGCTGATGAACAATCTCTTGCTCCTAAGTATTATTTCTATGATCCTAATTTCCTTATGGCTGGTTTCTCAGATATTACTAGATTCTCCGAAGCTCTTAAGTATCATTTTGTTAATCTTCAGTTCGCTGGTAATTATAAGGCTTTCGGTACTGCTGAAGCTCCTCAATTCAATCTTGTTCTTATTGATACTACCGCTATGGCTGATGCTCATGTTGATGGTGGTATTACTGTGAAACAACTTTCTGAGGGTGTTGATTTGACTAATGTTTTTGATTCTACCAAGAAGGCTAGGGTTGTTACCGAAGCAGCTGCTAGGTTGGCTGAAGATCAGCAGTTGAGAACTGGACTTACAATTATCGAAGCTGATCAGTGGGCTAACAGATGGCAGGATGCTGTTATCAGACTTAGACTTAAGGAAGGACTTTCTGGTGAGTGGATGCCTTTGCTTGAAAAGATGCAAGAAAATGCAGATGGTAGTTATCAGATTGATTTCATTAACACTAAGAACACTGGTGAAATTAGAGGTGTGTCAACTAGAGATGCTACTTTTAAGGAATTTAAGTCTTACCTTGATAAGAACCTTGATGCTTTGCATAAGGATTTCATTTTCCAGGATGGAGTGTTCAAACCTTTGGAGGGTGAGCCTATTGATGGTTTGAATGCTATGTTCGCTGTTCAGATGATTATTGATTGGTTCAATAGAGGTACTAACGCTGAGGAAGATGCTGCTTCTGGTTCTACTAATGCTCAGTTGTCTCTTGCTATTAAGGTTCATTCTTATATCAACATGGCTCAAGTGGCTCACACTGCTGTTACAGATGTTGCTAAGGTTTATTCTCTTGTTAAGTCTGCTCTTTACGGTGAACAAGTTGCTGGTGGTGCTTCTCTTCTTACTTTCGGTAAGGCTCTTAAATTCGCTGCTACTGAAGGTTTGGGTGTTTTGTTGGGTGCTTCTTTCGTTATTCTTGATGCTTATCAGCTTTCTCTTGTTGAAAATGATGTTCAAAAGGCTATTATCGGTACTCAGTTGGCTTTCGATTCTGCTTCTTTTTTGGTTTCTATTGGTGCTGCTGTTGCTGGTGTTTCTGGTGTTGGTATTGTTCTTTCTGGTGCTGGTGTTCTTTTGGCTGGTTTGGGTGTGGGTTTTGGTGCTTTGGCTCAGGCTTTTGGTCAGATTACTGCTGATGCTCAGTTGGTTGGTAAGTATTTTGCTTTGTTGGATGAGGCTTATTCTGGTGGTGGTTATAGGTATGAGTCTAAGGATAAGGCTTTGATGCCTTTGGCTGGTGCTGTGATTGATAAGATTAGACTTGCTGATAAGCATTTGCAAGTTCATTTTGATTCTCAATTCATCTACAGGACTCATCATGGTTCTACTGGTTCTGGTAAGATTAATTATTTCTTCTGGGCTGGTGATATGCCTCAAATGGTTCATGATAGATCTCAAGCTATTAATGTTAGGGAGCAGATTGGTTATAAGAATGATTCTGCTACTTTGGAATTGCCTGAAGATGCTTCTGATTTGATTTTGCCTGCTACTCCTAAGTCTTTCATTACATATAGATACGAGATCTTGCCATTTGCTACTTCTAGGCATGATAGAGGTTTTGATGTTATTAGAAGATTGGAAGAAGATTACAGGTTCGATTATGATTTCTACATTTTCCCTTCCGAATATACTATTAGGACTATTAAGCATGAGTACGTTGAAACTAAGGTGGCTGTTGTGTTGGATAGAAGATCTTTGAGATTGGGTGCTCCTGAGCTTTTGAAGGAGCAGCATGGTTATCTTATTTATGAAATTGAGGGTACTGGTGGTCAATATGTGGTTTGTTTGAATCATGGTAGTACTGTGACTTTGAAGACTACTGGTTCAGAAGCAAATAAGACACTTTGGATTTTGGATGCTAGACAGTTGATTGCTGGTAATGATAGTATTACTGTTTCTCAAGATTCCATTACTATCGGATCTACTACTGTTCACCTTAGAAATCAAGATTTCGATTGCCTTCAAATTGTTATGAGTAGAGAAGAGCTTTTGCAGGTGGATCTTGCTTCAAGACAGGCTAGGGTTTTGTCAGAAGATGCTTCAAAATGGCTTGATACAAAGGAAACTATCCATGAACACCTTCACAAGTTGGCTGAAGAACATAGACTTCTTGGTCAATTCGTGGTTGTGAATAATTATAGTGTTCAAGAACAGAGTGATATCGGTACTCTTTACAGAGGAGTTGGTAGAGCTTATTACGATGCTGCTCATGATAGAATGCTTTATACTGATGTTGAGGATGAGACTTTGACTAGGGATGCTGTTTTGGCAGGTGTGACTGGTGAGGATGCATATTTTTATAATGTGAAACAGAACGCTCTTTGGAGAGTTAATGCTGGTACTCACCAAGTTATTACTAAGTATTATCCTACAGTGTTGGAAGATCCTGATTCTAAGATGGCTAGAGTTGAACAGCATGGTAATATTGTTGTTATGGCTATGTCTAGGTTGAATCAACCTGATAAGGGTGTTGATGGAATTATTTATTTGGTGCATCAAGAATCTATGGTGTTGACTGCTGTTATTGGTAATTCTGAGATGCTTAAGAGACTTAGACAAACTTCAACTATTTCTCTTACTGAATTGAGGGGTGTGAACGATATGGCTCTTGTTTCTACTATGGATAAGCTTCCAGGTCAGAAGGTGGTTGCTACTAACGCAGAATTCGTTACAATTTTGGCTAAGGATGAACAAGGAAGCCAGGTTTGTTTTTGGCTTAGGACTGTTGATGGTGTTTTGATTAAGCCTTCCATTCAACCTCCTATTCCTACAGATCTTGCTCTTACTGTTTCTTCTCATAAAGTTGCTGGTCAAGATGTTTTTTATTTCTGTTCTATCAAGGAGCAGACTGTTTATAGACAACAAGGTACCGGACAAACCAACGATACATCTGTTGGTTCTATTCCTATCCAAGGTCTTTCTAATCTTTTCACTGTTTATGGTGAGGTTTTCGCTACTACTGATGATGGTAGGATTCTTAGATTGCTTGGTGATGGTTCTACTTATCTTGAGGGTATTAACGAACATTGGTTGATTAAGCATCCTAAGTGGTGGCAAGATTTGGCTACTCTTGAAGCAGAGACTGCTACTACTATTGCAGTTTTCGGTGTTAGGGATCCTCAAAATGCTGTTGTTCCTATTTGGTATCATTCCGGTAAGATTGCTGTTGCTTCTTCTAAGCTTTATGGTAAGCAGCTTCAATTTCTTGGTTTCCATTCTAATGATCAAGCTTGGCTTTTCGATCTTGATGGTGGTATCTTGTATTCTCAACCTCTTGTTGATAGGCAAACTGTTGATACTATTTTCTCCCTTGATGGTAAGCTTATTGCTCCTGATTTGATTCCTCAAGTTCAACAAATGCTTTCTGGTCAGCACCTTAGAAACCCTGTTATTGTTGATGGTAGACTTCATGTGACTACCATTGATGGTGTTGTTGTGGGTTTCGATAAGAAGAGCCCTACTCTTGTTGCTGTGAATAAGGCTTGGCAAATTTCCAGGCAAAATAGGTTGGAGGAGGCTCTTGGTAATTTGCTTTCTAATTGGTCTGATCATAACGATGCTATTGTTCTTCAAGGTGATCCTGAGTCTGCTCCTGCATGGTATTACGTTCCTTCTAGAGAAATTGTGACTGCTAATGGTCTTACTTGGGCTGATCATCCTTTGTGGCTTGGTATGGCTGTTGGTGCTGCTACTCCTTATATTTATAATACTTCTCCTAGGAGGAGGTCTATTGTTCCTGAAGAGATTACTATCCCTACTCTTGCTGGTATTGATTTCGCTTTGGTTGCTGCTACTAATGCTACTATTGTTATTGATAGAGATAACTGGAAGCATTATAAGGCAATTGTTCTTGATAACAAGGTTGATCAAGATGGAGGTGCTAAGGTTGTGTTGGGTTCTGAGATTCCTATGAAGGTTTTGGTTAGAAAGTTTCAAGGTAATTTGGAATTCTTCGATCCTAATAATGGTAATGTTGTGATTTTGCAAAACGTTCTTAAGGGTGATGGTGCTTGTTGTCAGATTGATATTAAAGTTGGTGATGCTGAAACTATGCTTGCTTCTGATTTCTCTGAGATTTTCTCCAAGACCCATCCTCTTGTTGCTTCTCAGGTTTTCGAATTGGGTGATGTTATTGCTGTTACTAAGCATAAGTCTGATTATGAGGATCTTTGGGCTATTTTGCATATGACTTGA
the invention provides a protein for coding the insect-resistant cyhalothrin gene and homologous genes thereof, wherein the amino acid sequence of the protein is as follows:
SEQ ID NO.11
Met Val Met Val Ile Asp Tyr Pro Ser Leu Arg Ala Ser Leu Tyr Ala Thr Lys Ser Ala Val Pro Cys Arg Pro Leu Pro Gly Asn Pro Glu Arg Val Ser Arg Arg Val Ser Arg Arg Gly Ala Gly Glu Ile Gly Ser His Ala Asp Phe Trp Ile Ser Arg Ala Lys Ala Ser Ala Ile Arg Ile Phe Pro Arg Gln Glu Gly Pro Leu Arg Glu Ser Arg Arg Arg Phe Tyr Leu Leu Arg Glu Ala Val Phe Thr Gly Arg Arg Thr Ala Trp Ala Ala Asn Pro Ala Ala Gln Arg Ala Ala Trp Asp Arg Lys Arg Ser Pro Phe Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu Glu Phe Leu Thr Gln Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr Ala Gln Thr Val Thr Lys Ser Pro Val Leu Pro Ala Gln Gly Trp Val Lys Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Phe Val Glu Leu Val Ala Glu Lys Ser Asp Glu Thr Val Lys Ala Tyr Leu Leu Gly Tyr Asn Gly Lys Asn Asp Ser Pro Glu Pro Ala Tyr Val Asp Ile Pro Lys Arg Gly Ala Glu Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu Pro Arg Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg Ala Leu Gln Asp Glu Leu Lys His Phe Ala Asn Thr Leu Glu Val Asp Pro Asp Ile Val Ser Asn Ala Val Asp Gly Glu Tyr Glu Gly Gly Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala Ser Met Arg Gln Lys Met Ala Glu Arg Lys Lys Leu Leu Gln Thr Lys Lys Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr Lys Phe Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr Asp Asn Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp Leu Glu Ile Lys Lys Ala Lys Gly Thr Asn Ala Val Val Asn Ser Gly Ala Ala Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu Met Glu Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln Gly Tyr Glu Ser Tyr Gln Thr Val Arg Val Pro Gly Tyr Asn Glu Asp Met Thr Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr Asp Glu Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp Gln Glu Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu Val Arg Val Met Ala Val Ala Leu Ser Glu Gly Glu Asp Val Ala Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Val Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile Asp Thr Thr Ala Met Glu Asp Ala His Val Asp Gly Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys Ser Tyr Leu Asp Lys Asn Leu Asp Val Leu His Lys Asp Phe Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Glu Thr Ser Leu Phe Thr Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser Ala Ser Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser Gly Val Gly Met Ile Leu Gly Gly Ala Gly Val Val Leu Gly Gly Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Arg Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met Pro Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile Gly Tyr Lys Asn Asp Asn Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Thr Gly Phe Asp Val Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly Gly Gln Tyr Val Ile Cys Leu Asn Gln Gly Ser Thr Val Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln Val Asp Leu Ala Ser Gly Glu Ala Arg Val Phe Ser Glu Asp Ala Ser Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Glu Glu His Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr Arg Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Leu Leu Glu Asp Pro Asp Ser Asn Met Ala Arg Val Glu Gln His Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp Lys Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Ile Val Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Arg Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Leu Leu Ala Lys Asp Glu Gln Gly Ala Asn Ile Lys Glu Gln Thr Val Tyr Cys Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Ile Glu Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Leu Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asn Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Cys Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr Thr Val Ala Val Phe Gly Val Arg Asp Leu Gln Asn Ala Val Val Pro Ile Trp Tyr His Asn Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys Gln Leu Gln Phe Leu Gly Phe Asn Ser Asn Gly Glu Ala Trp Leu Phe Asp Leu Asp Asp Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser Phe Leu Tyr Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Ile Phe Ile Pro Gln Val Glu Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Ser Thr Ser Arg Arg Arg Val Tyr Val Val Glu Ser Lys Asn Met Ala Lys Asp Lys Gly Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Ile Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile Asp Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Val Gly Ser Glu Ile Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Cys Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr Ala Asp Leu Trp Ala Ile Leu Arg Met Thr*
SEQ ID NO.12：
Met Glu Glu Ala Asp Met Glu Asn Arg Gly Tyr Glu Glu Arg His Ser Gln Ser Gln Ser Ala Thr Pro Leu Arg Arg Arg Lys Arg Ser Pro Phe Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu Glu Phe Leu Thr Lys Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr Ala Gln Thr Val Thr Lys Ser Pro Val Leu Pro Ala Gln Gly Trp Val Lys Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Ser Gly Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu Pro Arg Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg Ala Leu Gln Asp Glu Leu Lys His Phe Ala Asn Thr Leu Glu Val Asp Pro Glu Ile Val Ser Asn Ala Val Asp Gly Glu Tyr Glu Gly Gly Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala Ser Met Arg Gln Lys Met Ala Glu Arg Lys Lys Arg Leu Glu Arg Asn Lys Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr Lys Phe Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr Asp Asn Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp Leu Glu Ile Lys Lys Ala Lys Gly Thr Asn Ala Val Val Asn Ser Gly Ala Ala Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu Met Glu Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln Gly Tyr Glu Ser Tyr Gln Thr Leu Arg Val Pro Gly Tyr Asn Glu Asp Met Thr Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr Asp Glu Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp Gln Glu Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu Val Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Val Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Ile Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Thr Ala Ala Arg Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys Ser Tyr Leu Asp Lys Asn Leu Asp Val Leu His Lys Asp Phe Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Glu Ala Ser Leu Leu Thr Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser Ala Ser Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser Gly Val Gly Met Ile Leu Gly Gly Ala Gly Val Val Leu Gly Gly Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Arg Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Leu Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met Pro Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Ile Arg Glu Gln Ile Gly Tyr Lys Asn Asp His Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Gln Tyr Gln Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly Gly Gln Tyr Val Ile Cys Leu Asn Gln Gly Ser Thr Val Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln Val Asp Leu Ala Ser Gly Glu Ala Arg Val Phe Ser Glu Asp Ala Ser Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Glu Glu His Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr Arg Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Leu Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp Lys Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Lys Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Ile Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro Ile Pro Thr Asp Leu Thr Leu Thr Val Ser Ser His Lys Val Ala Gly Gln Asp Val Phe Tyr Phe Tyr Ser Ile Lys Glu Gln Thr Val Tyr Cys Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Ile Glu Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Leu Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asp Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Tyr Leu Asp Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr Thr Val Ala Val Phe Gly Val Arg Asp Leu Gln Asn Ala Val Val Pro Ile Trp Tyr His Asn Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys Gln Leu Gln Phe Leu Gly Phe Asn Ser Asn Gly Glu Ala Trp Leu Phe Asp Leu Asp Asp Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser Phe Leu Tyr Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Ile Phe Ile Pro Gln Val Glu Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Ser Thr Ser Arg Arg Arg Val Tyr Ala Val Glu Ser Lys His Met Ala Lys Asp Lys Gly Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Ile Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile His Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Val Gly Ser Gly Ile Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn Val Val Ile Leu Arg Asn Val Leu Lys Gly Asp Gly Ala Cys Ser Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr Ala Asp Leu Trp Ala Ile Leu Arg Met Thr
SEQ ID NO.13：
Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Ala Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Arg Met Gln Glu Asn Gly His Gly Ser Tyr Gln Ile Glu Phe Leu Asn Thr Lys Asn Thr Gly Glu Thr Arg Trp Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys Ser Tyr Leu Asp Lys Asn Phe Asp Ala Leu Arg Lys Asp Phe Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Asp Gly Glu Pro Ile Asp Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Pro Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Glu Ala Ser Leu Leu Thr Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser Ala Ser Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser Gly Val Gly Met Ile Leu Gly Gly Ala Gly Val Ile Leu Gly Gly Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Gln Ile Thr Ala Asp Ala Gln Leu Val Gly Glu Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Ser Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys Arg Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Val Gly Asp Met Pro Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile Gly Tyr Lys Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly Gly Gln Tyr Val Ile Cys Leu Asn His Gly Ser Thr Val Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe Asp Cys Leu Gln Ile Val Met Ser Arg Asp Glu Leu Leu Gln Val Asp Leu Ala Ser Arg Glu Val Arg Val Phe Ser Glu Asp Ala Ser Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Gly Glu His Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr Arg Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Asn Gln Asn Ala Leu Trp Arg Val Asn Ala Asp Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Leu Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp Lys Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Arg Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Leu Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro Ile Pro Thr Asp Leu Thr Leu Thr Val Ser Ser His Lys Val Ala Gly Gln Asp Val Phe Tyr Phe Tyr Ser Ile Lys Glu Gln Thr Val Tyr Cys Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Ile Glu Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Leu Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asn Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Tyr Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr Thr Val Ala Val Phe Gly Val Arg Asp Pro Gln Asn Ala Val Val Pro Ile Trp Tyr His Asn Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys Gln Leu Gln Phe Leu Gly Phe Asn Ser Asn Gly Glu Ala Trp Leu Phe Asp Leu Asp Asp Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser Gln Ala Val Asp Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Asn Leu Ile Pro Gln Val Glu Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Leu Tyr Ile Tyr Ser Thr Ser Arg Arg Arg Val Tyr Ala Val Glu Ser Lys Lys Met Ala Lys Asp Lys Gly Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Thr Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile Asp Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Val Gly Ser Glu Ile Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Ser Lys Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr Ala Asp Leu Trp Ala Ile Leu Arg Met Thr
SEQ ID NO.14：
Met Glu Glu Val Asp Met Gln Asn Arg Gly Tyr Glu Glu Trp His Ser Gln Ser Gln Ser Ala Thr Thr Leu Arg Arg Arg Lys Arg Ser Pro Val Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu Glu Phe Leu Thr Gln Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr Ala Glu Thr Val Thr Lys Ser Pro Val Leu Pro Glu Arg Gly Trp Val Lys Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Phe Val Gln Val Val Ala Glu Gln Ser Tyr Asp Thr Val Lys Ala Tyr Leu Leu Gly Tyr Asn Gly Glu Asn Asp Ser Pro Asn Pro Ala Tyr Val Asp Ile Pro Lys Arg Ala Ala Glu Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu Pro Arg Gln His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg Ala Leu Gln Asp Glu Leu Lys Lys Phe Ala Asp Met Phe Gly Val Asp Pro Glu Ile Val Ser Asn Ala Val Asp Gly Lys Tyr Lys Gly Gly Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala Ser Met Arg Gln Lys Met Ala Glu Arg Asn Lys Val Leu Leu Thr Met Lys Leu Glu Gln Pro Arg Ser Ser Arg Val Leu Pro Leu Val Asn Ala Thr Gln Arg Trp Arg Arg Pro Phe Gly Cys Val Ala Thr Gln Arg Arg Leu Ser Lys Ala Gly Arg Leu Val Gln Ala Tyr Ile His Arg Asp Gln Glu Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu Val Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Lys Asn Pro Glu Tyr Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Ala Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr Lys Pro Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile Asp Thr Thr Val Met Ala Asp Ala His Val Asp Gly Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Gly Thr Phe Lys Glu Leu Lys Ser Tyr Leu Asp Lys Asn Leu Asp Ala Leu His Lys Asp Phe Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Ser Leu Val Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Gly Ala Ser Leu Leu Thr Phe Gly Lys Ala Leu Lys Leu Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala Pro Val Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Asp Phe Asp Ser Ala Ser Phe Leu Val Ser Ile Gly Ala Ala Val Ala Gly Val Ser Gly Val Gly Ile Val Leu Ser Gly Ala Gly Val Leu Leu Ala Gly Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Arg Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Val Gly Asp Met Pro Lys Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile Gly Tyr Lys Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile Arg Arg Leu Glu Glu Asp His Arg Phe Asp Tyr Val Phe Tyr Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val Ala Val Val Pro Asp Arg Arg Pro Leu Arg Leu Arg Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly Gly Glu Tyr Val Ile Cys Leu Asn Lys Gly Ser Thr Val Thr Leu Phe Thr Thr Gly Ser Glu Ala Asn Lys Thr Ser Trp Ile Leu Asp Ala Arg Gln Ile Ile Ala Gly Asn Asp Pro Ile Ile Val Ser Gln Asp Ser Ile Thr Met Gly Ser Thr Thr Val His Leu Arg Asn His Asp Phe Asp Cys Leu Arg Ile Val Thr Ser Arg Glu Glu Leu Leu Gln Val Asp Ser Ala Ser Arg Gln Thr Arg Val Leu Ser Glu Asp Ala Ser Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Glu Glu His Arg Leu Leu Gly Gln Phe Val Val Val Asn Asn Tyr Ser Val Gln Glu Gln Ser Asp Ile Gly Thr Leu Tyr Arg Gly Val Gly Arg Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Val Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln Arg Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Ser Asp Lys Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Ala Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Lys Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Ile Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro Ile Pro Thr Asp Leu Thr Leu Thr Val Ser Ser His Lys Val Ala Gly Gln Asp Val Phe Tyr Phe Tyr Ser Ile Lys Glu Gln Arg Val Tyr Arg Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Val Gly Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Phe Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asn Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Tyr Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr Thr Ile Ala Val Phe Gly Val Arg Asp Pro Gln Asn Ala Val Val Pro Ile Cys Asn Gly Glu Ala Trp Leu Phe Asp Leu Asp Gly Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser Gln Ala Val Asp Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Asn Leu Ile Pro Gln Val Gln Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Gly Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Arg Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Ser Thr Ser Arg Cys Arg Val Tyr Ala Val Glu Ser Lys Asn Met Ala Lys Asp Lys Gly Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser Ile Val Pro Ala Glu Ile Thr Ile Pro Met Leu Ala Gly Ile Asp Phe Ala Leu Val Ala Ala Thr Lys Ala Thr Ile Val Ile Asp Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Val Gly Ser Glu Ile Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Ser Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe Glu Leu Gly Asp Gly Cys Asp Cys Ser His Lys Thr Gln Ile Gly Leu Cys Gly Ser Val Gly His Ser Thr His Asp Ile Ala Tyr Ile Ser His Ala Tyr Leu His Arg Trp Pro Ile Tyr Val Ile Ser Thr Leu Ile Ala
SEQ ID NO.15：
Met Glu Glu Ala Asp Met Glu Asn Arg Gly Asn Glu Glu Arg His Ser Gln Ser Gln Ser Ala Thr Pro Leu Arg Arg Arg Lys Arg Ser Pro Val Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu Glu Phe Leu Thr Arg Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr Ala Glu Ala Val Thr Lys Ser Pro Ala Leu Pro Ala Gln Gly Trp Val Lys Val Gly Arg Val Pro Gly Arg Gly Asp Val Leu Phe Val Gln Leu Val Ala Glu Lys Ser Asp Glu Thr Val Lys Ala Tyr Leu Leu Gly Tyr Asn Gly Glu Asn Asp Phe Pro Lys Pro Ala Tyr Val Asp Ile Pro Lys Arg Gly Ala Lys Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Asp Pro Arg Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg Ala Leu Gln Asp Glu Leu Lys Lys Phe Ala Asp Met Phe Gly Val Asp Pro Glu Ile Val Ser Asn Ala Val Asp Gly Lys Tyr Glu Gly Gly Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala Arg Met Arg Gln Lys Met Ala Glu Arg Lys Lys Leu Leu Glu Thr Lys Lys Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr Glu Phe Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr Asp Asn Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp Leu Glu Ile Lys Lys Ala Arg Gly Thr Asn Ala Val Val Asn Ser Gly Ser Ala Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu Met Glu Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln Gly Tyr Glu Ser Tyr Gln Thr Val Arg Val Pro Gly Tyr Asn Glu Asp Met Thr Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr Asp Glu Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp Gln Glu Asn His Leu Leu Arg Gln Lys Thr Gln Glu Ile Glu Asn Thr Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu Val Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Val Ala Val Ile Ala Asp Glu Gln Ser Leu Ala Pro Lys Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Arg Leu Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys Ser Tyr Leu Asp Lys Asn Leu Asp Ala Leu His Lys Asp Phe Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Ser Leu Val Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Gly Ala Ser Leu Leu Thr Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser Ala Ser Phe Leu Val Ser Ile Gly Ala Ala Val Ala Gly Val Ser Gly Val Gly Ile Val Leu Ser Gly Ala Gly Val Leu Leu Ala Gly Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Gln Ile Thr Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Ser Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met Pro Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile Gly Tyr Lys Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly Gly Gln Tyr Val Val Cys Leu Asn His Gly Ser Thr Val Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln Val Asp Leu Ala Ser Arg Gln Ala Arg Val Leu Ser Glu Asp Ala Ser Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Glu Glu His Arg Leu Leu Gly Gln Phe Val Val Val Asn Asn Tyr Ser Val Gln Glu Gln Ser Asp Ile Gly Thr Leu Tyr Arg Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Val Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn Gln Pro Asp Lys Gly Val Asp Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Lys Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Ile Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro Ile Pro Thr Asp Leu Ala Leu Thr Val Ser Ser His Lys Val Ala Gly Gln Asp Val Phe Tyr Phe Cys Ser Ile Lys Glu Gln Thr Val Tyr Arg Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Val Gly Ser Ile Pro Ile Gln Gly Leu Ser Asn Leu Phe Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asp Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Tyr Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Thr Ala Thr Thr Ile Ala Val Phe Gly Val Arg Asp Pro Gln Asn Ala Val Val Pro Ile Trp Tyr His Ser Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys Gln Leu Gln Phe Leu Gly Phe His Ser Asn Asp Gln Ala Trp Leu Phe Asp Leu Asp Gly Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Arg Gln Thr Val Asp Thr Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Asp Leu Ile Pro Gln Val Gln Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Asn Thr Ser Pro Arg Arg Arg Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Ile Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile Asp Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Leu Gly Ser Glu Ile Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Cys Gln Ile Asp Ile Lys Val Gly Asp Ala Glu Thr Met Leu Ala Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr Glu Asp Leu Trp Ala Ile Leu His Met Thr
example 2: construction of plant expression vector of 35s promoter + cycotoxin gene
Preparation of a linearized vector: the VM062-3 FLAG vector was double digested with restriction enzymes BstBI and MluI to linearize the circular vector.
Amplification of target sequences with homology arms:
SEQ ID NO.17：
with the forward primer:
5’-gggcggtaccccgggttcgaaATGGTTATGGTAATTGACTATCCAAGTC-3’；
reverse primer:
5'-cactagttaattaagacgcgtCTATGTCATGCGTAGAATGGCCC-3' is a primer, PCR amplification is carried out to the cycadin gene, the amplification system is:
KOD One TM PCR Master Mix 25μL
upstream primer (10. mu.M each) 1.5. mu.L
Downstream primer (10. mu.M each) 1.5. mu.L
Template 2. mu.L
20 mu L of sterilized water
Total volume 50. mu.L
The PCR program is 98 ℃ for 5 min; 30 cycles of 98 ℃ for 30s, 50 ℃ for 30s and 68 ℃ for 3 min; then storing at 68 deg.C for 10min and 4 deg.C.
And (3) detecting and recovering a vector enzyme digestion product and a PCR product: agarose gel electrophoresis is used for detecting PCR products and carrier enzyme digestion products
Figure BDA0003571196030000601
The Quick Gel Extraction Kit recovers the target fragment.
Glue recovery product recombination: use of
Figure BDA0003571196030000611
Basic Seamless Cloning and Assembly Kit for homologous recombination, reaction system as follows:
2×Basic Assembly Mix 5μL
enzyme digestion vector X mu L
PCR product Y uL
Sterilized water to 10 μ L
In a 10 μ L reaction system, the addition of the vector and each insert is preferably 0.01 to 0.25pmols, and the optimal molar ratio of vector to each insert is 1: 2.
mix gently and react at 50 ℃ for 15 minutes.
The transformation method comprises the following steps:
1. thawing the Trans1-T1 competent cells on ice, standing for 2-5 minutes, observing whether the cells have thawed, flicking the bottom of the tube 1-2 times with a finger.
2. mu.L of the recombinant product was added to 50. mu.L of the cells, and the tube walls were gently flicked and centrifuged to mix well, followed by placing on ice for 30 minutes.
Heat shock was applied to the water bath at 3.42 ℃ for 30 seconds, followed immediately by cooling on ice for 2 minutes.
4. After adding 450. mu.L of a normal temperature non-resistant LB liquid medium, the mixture was incubated at 37 ℃ for 1 hour with shaking at 250 rpm.
The cells were centrifuged at 5.5000 rpm for 2 minutes, collected, 100. mu.L of the liquid was resuspended, and evenly spread on LB solid plates containing kanamycin resistance, followed by overnight culture in an incubator at 37 ℃.
Sequencing and identifying: positive clones are screened by colony PCR and plasmid PCR, and are sent to Shanghai biological engineering Co., Ltd for sequencing analysis, and the results show that: the cyathrin gene plant overexpression vector VM062-Suit (shown in figure 2) is successfully obtained.
The invention selects VM062-3 FLAG vector as basic plant expression vector, because the enzyme cutting site between 35S promoter and 3 FLAG is easy to generate frame shift mutation, and the label is positioned at the N end of protein to be beneficial to protein expression, the invention adopts BstBI and MluI enzyme cutting site behind 3 FLAG label to construct final expression vector. The vector sequence is shown as sequence SEQ ID NO. 18.
SEQ ID NO.18 plant expression vector sequence:
GAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGTCTACCATGGCTCAGATCAGGTCGATGGCTCAGGGCATTCAGACGCTGTCGCTCAACTCCTCTAATCTCTCCAAGACGCAGAAGGGGCCGCTCGTGTCGAACTCTCTCTTCTTCGGCAGCAAGAAGCTGACACAGATCTCTGCCAAGTCACTGGGGGTTTTCAAGAAGGACTCGGTGCTCCGGGTGGTCCGCAAGTCCAGCTTCCGCATCTCAGCTTCCGTCGCTACAGCTGAGGCTGAGGCCGTGATCGCTGAGGTCTCCACTCAGCTCAGCGAGGTTGTGGGCGTGATCGAGAGGCACCTGGAGCCAACCCTCCTGGCTGTCCATCTCTACGGGTCAGCGGTTGATGGTGGCCTGAAGCCCCACTCCGACATCGATCTCCTGGTTACAGTGACTGTCCGGCTGGACGAGACCACGAGGAGGGCTCTCATTAACGATCTCCTGGAGACCAGCGCTTCGCCAGGCGAGTCCGAGATCCTCAGGGCGGTTGAGGTGACGATTGTCGTTCATGACGATATCATTCCATGGAGGTACCCAGCTAAGAGGGAGCTCCAGTTCGGCGAGTGGCAGCGCAATGACATCCTGGCCGGGATTTTCGAGCCAGCGACAATCGACATTGATCTGGCTATCCTCCTGACTAAGGCTAGGGAGCACTCCGTCGCTCTGGTTGGCCCTGCTGCTGAGGAGCTCTTCGACCCAGTCCCTGAGCAGGATCTCTTCGAGGCCCTGAACGAGACCCTCACGCTGTGGAATTCTCCGCCCGACTGGGCTGGCGATGAGAGGAATGTGGTCCTCACCCTGTCGCGCATCTGGTACTCTGCTGTCACGGGGAAGATTGCTCCAAAGGACGTGGCTGCTGATTGGGCGATGGAGAGGCTGCCAGCTCAGTACCAGCCTGTGATCCTCGAGGCTAGGCAGGCTTACCTGGGCCAGGAGGAGGACAGGCTCGCGTCCCGGGCTGATCAGCTGGAGGAGTTCGTTCATTACGTCAAGGGCGAGATTACTAAGGTCGTTGGCAAGTGACTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAAGCTTGCATGCCTGCAGGTCCCCAGATTAGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTAGAGGATCGCGAGGCGCGCCGTCGACATGGACTACAAGGACGACGATGATAAGGGCATGGACTACAAGGACGACGATGATAAGGGCATGGACTACAAGGACGACGATGATAAGGGCGGTACCCCGGGttcgaaATGGTTATGGTAATTGACTATCCAAGTCTCAGAGCTTCGTTATATGCCACTAAGAGTGCGGTCCCATGTCGCCCGCTTCCGGGCAATCCAGAGCGGGTGTCGCGCCGGGTATCACGCAGAGGCGCGGGAGAGATCGGGTCCCATGCAGATTTCTGGATCTCCCGCGCCAAGGCATCCGCCATCCGGATTTTCCCGCGTCAAGAGGGTCCGCTCCGTGAATCCCGGAGAAGATTCTATCTTCTCCGCGAAGCGGTGTTCACCGGCCGCAGGACCGCCTGGGCCGCTAATCCAGCGGCCCAACGGGCAGCATGGGACCGCAAGAGATCCCCTTTCTTCGGAAGGTCTTCCGATGATATTCATTCATTCAGCAAGAATCCAGAAGAATTCTTGACTCAAAACACTCTCAGCTGCAGTTTATTAACTCGCACCGCTCAGACCGTCACAAAAAGCCCAGTATTGCCCGCCCAAGGTTGGGTCAAAGTGGGTCGTGTGCCAGGAAGAGGAGACGCTCTCTTTGTTGAGCTGGTTGCCGAGAAATCAGATGAGACAGTGAAGGCGTACTTACTTGGCTACAATGGTAAGAACGACTCCCCCGAGCCGGCATACGTTGATATTCCAAAGAGGGGCGCCGAGAACGGAACATTTTTGTTTACCGTGGATCTCTCGGGCTGCTCCGTTGTTGTGACACAGTTGGACGAGCAGACATACCGTGTTTTCCACGACAAACGGGTGAACAGCTCCATTCTGTACGACAACGTCGTAATGTATGTGGATTTCAATCACTACCGCTATCCCAGAGCCCACGGACACGGGAGAGATACGGGACAAGCGGTCGTCTGTATGCAATTCGCCGACGGAGTGTGGAAATTGTTTCTCCAGCGGCACGAGACGGCGAATTCTCTCCCAAAGATATGGTCCCGTGGCGATGGCAAACCGGAGATTTTCAGCGACGCCGAACCCCGTCCACATATGGAAGATGGCATAAAGGAATTTCTCAATCGGCGTCGAGCTTTGCAAGACGAATTGAAGCACTTCGCCAACACGCTTGAAGTGGACCCGGACATAGTGTCCAACGCTGTCGACGGCGAATACGAAGGCGGCCAGTTTTCCAGGAACGACGATGCAATTCGTGGATGGAACGAATTACGTGCAAGCATGAGACAGAAAATGGCTGAAAGGAAGAAACTGTTGCAAACAAAGAAAGGTATGCTAGACTCATTCAACAACCCGGATCACCCCCTCTATAAATTTCGCAAACAAGTGCTCGACGAGCTCAAGGAAATGAGTGCATACGACAACACCTTGGAGGCATCCGAGATTGCGGACCGTACTTGGCTGTGGTTGGAGATCAAAAAAGCCAAAGGTACGAATGCAGTGGTAAACAGTGGAGCAGCCCCGAAGCAGCCGAGTCTTACCGTTGGTGAACGCTACGCAGAGATGGAGGCGGCCCTTTGGATGCGTAGCGACACAGCGGCGTTTGAGCAAGGGTATGAATCTTATCAAACCGTTCGCGTGCCGGGTTACAATGAAGATATGACATCCATTGCAATGAAGAAGTTGTTCGTTGAGAGCAGTCTGACTGACGAGCAGCTGGGCGCCTTGTGCAAGCGTATATCCATTGCTAGCGATCAAGAGAACCATCTTCTGACACAGAAGACGCAGGAGATTGAGAGCACTTTCAAACAATTTTCAGATGGCAGCTTTTTGGAGTCGGCGCCTCAGGATTTCTTCCTGAGACGAGTCGGTGATAAATCTGGAGGACGCTGCTATCCTCTTGTAAGGGTAATGGCCGTCGCACTGTCTGAAGGAGAAGACGTTGCCCAGAATTTTATGGAGAACCTGAGGACATTGTCTAGGAATCCAGAGTATTCAGGCGAATCGGCAAGTTCACAAGCCACAAATGCAGGGTTGTTCAGAGATGCCCTGGCCGGCCTGCACGCGAATGTGGAAGCCATTGCCGCATCTGAGAAACTACCTGCTGTGTTTGGACTGCCCGATGTTGCTACAATGTTGAGCCAAGCGAGCGAAAGCAAGGTTTACGCTTTGAATACCGAAACACACAGCATGCTGGTTGCGGTGATTGTAGACAAACAGAGTCTGGCGCGCAAGTATTACTTCTATGATCCCAACTTTCTCATGGCCGGTTTCTCCGACATAACTCGTTTCTCGGAAGCCCTCAAGTATCACTTTGTGAATCTCCAGTTCGCCGGAAACTACAAAGCGTTTGGCACAGCGGAGGCTCCCCAGTTTAACCTGGTCCTTATTGATACGACCGCCATGGAGGATGCGCATGTTGACGGAGGGATCACCGTGAAACAATTGAGCGAGGGCGTTGATCTTACTAATGTCTTTGACAGCACCAAAAAGGCTCGTGTGGTAACTGAGGCAGCAGCTCGCTTGGCTGAAGACCAACAACTGCGCACTGGCCTCACCATAATAGAGGCTGACCAGTGGGCAAACAGGTGGCAAGATGCCGTCATACGATTGAAACTCAAGGAAGGATTAAGTGGAGAGTGGATGCCATTGCTGGAGAAAATGCAAGAAAACGCAGATGGTTCTTATCAGATTGATTTCATCAACACCAAGAATACTGGGGAAATCCGTGGGGTTTCCACTAGAGATGCGACGTTCAAAGAATTCAAATCCTACCTGGATAAAAATTTAGACGTATTGCATAAGGATTTCATCTTTCAGGATGGAGTTTTCAAACCACTGGAGGGAGAGCCCATTGACGGGTTGAATGCAATGTTCGCGGTCCAAATGATTATAGATTGGTTCAACCGGGGGACTAATGCTGAGGAAGATGCGGCCTCAGGATCCACCAATGCCCAACTTTCCCTAGCGATTAAAGTGCACAGCTACATTAACATGGCCCAAGTAGCGCATACTGCAGTTACAGATGTTGCCAAGGTATACAACCTTGTAAAGTCAGCACTGTATGGCGAGCAGGTAGCTGGGGAGACATCGCTGTTCACATTTGGGAAGGCGTTGAAATTTGCAGCAACAGAAGGGTTGGGAGTCTTACTAGGGGCGTCCTTTGTGATCTTGGATGCCTACCAGCTGTCCCTCGTGGAAAACGATGTGCAGAAGGCCATTATCGGCACACAACTAGCCTTTGATTCCGCAAGCTTTCTAGCGGGTATTGGTGCAATTGTTGCTGGAGCATCGGGCGTGGGCATGATTTTGGGCGGAGCTGGCGTGGTATTGGGAGGCCTGGGAGTTGGCTTTGGAGCCTTAGCTCAGGCATTCGGGGAAATCACAGCAGACGCCCAACTGGTGGGCAAGTACTTCGCACTCTTGGACGAGGCATACCGCGGTGGCGGATACAGATACGAATCTAAAGACAAGGCATTGATGCCTCTTGCAGGAGCAGTCATCGACAAAATACGACTGGCCGACAAACATCTCCAAGTTCATTTCGACAGCCAATTCATATATCGAACCCATCATGGCAGCACAGGATCCGGCAAAATTAATTACTTCTTCTGGGCTGGCGACATGCCGCAGATGGTCCACGATCGCTCCCAAGCTATCAATGTTCGAGAGCAAATTGGGTATAAGAACGATAATGCTACTTTGGAGCTACCGGAGGATGCCTCTGATCTGATCCTGCCTGCCACGCCCAAGTCATTTATCACTTACCGATACGAGATTCTACCATTCGCTACTTCCCGCCACGATACGGGTTTCGATGTCATTAGACGCCTGGAAGAAGACTACAGATTCGATTACGATTTCTACATCTTCCCATCTGAATACACAATCAGAACCATCAAACACGAGTACGTAGAAACAAAGGTGGCAGTAGTCCTGGATAGGCGCTCTTTGCGACTGGGAGCACCTGAGCTTCTCAAGGAGCAACATGGCTATTTGATCTATGAGATCGAGGGGACCGGAGGACAATACGTTATATGTCTTAACCAGGGATCCACTGTCACACTGAAGACCACCGGATCAGAGGCAAATAAAACATTATGGATACTTGATGCCCGACAACTCATTGCAGGCAACGATTCAATCACGGTCTCTCAAGATAGCATCACCATCGGCAGCACAACAGTCCATCTCCGTAACCAAGATTTCGATTGCCTACAGATCGTCATGAGTCGGGAGGAGCTTCTCCAAGTGGACTTGGCGTCTGGAGAAGCCCGCGTGTTTTCAGAAGATGCCAGTAAATGGCTCGACACCAAAGAAACAATCCATGAACATCTCCATAAGTTGGCTGAAGAACACAGGTTGCATGGCCAGTTTGTAGTGGTGAACAATTATTATCGTGGAGTGGGCCGTGCCTACTATGATGCTGCTCACGATCGGATGTTGTATACAGATGTGGAAGATGAAACTCTGACTCGGGATGCTGTTCTTGCCGGCGTTACAGGCGAAGATGCCTACTTCTACAATGTAAAGCAGAATGCCCTGTGGCGAGTGAATGCAGGCACACACCAAGTAATAACCAAATACTATCCGACGTTACTGGAGGACCCTGACAGCAACATGGCAAGAGTTGAGCAGCATGGCAACATCGTCGTGATGGCCATGAGCAGGCTCAACCACCCGGACAAGGGAGTAAATGGAATCATCTATCTCGTTCATCAAGAAAGCATCGTGCTTACTGCTGTGATCGGCAATTCAGAGATGCTCAAACGTCTTCGCCAAACGAGTACAATTAGCTTAACAGAATTGCGAGGTGTCAATGACATGGCTCTGGTTTCCACGATGGACAGATTACCCGGCCAAAAGGTTGTCGCTACCAACGCTGAGTTTGTGACACTTCTGGCTAAGGATGAGCAAGGAGCCAATATCAAGGAGCAGACGGTCTATTGCCAACAAGGTACGGGCCAGACCAACGATACATCTATTGAAAGCATCCCGATTCCAGGGCTCTCTAATCTTTTAACCGTGTACGGTGAAGTATTCGCTACAACTAATGATGGTCGCATCCTTCGCCTGCTTGGGGACGGCTCAACATGTCTCGAGGGCATCAATGAGCATTGGCTCATCAAGCATCCAAAATGGTGGCAGGACTTGGCTACCTTGGAGGCAGAGAAAGCCACGACAGTTGCAGTATTCGGTGTTAGAGACCTACAAAATGCAGTGGTGCCAATTTGGTACCACAACGGAAAGATCGCGGTGGCCTCATCAAAACTGTATGGCAAACAACTGCAATTTTTGGGGTTTAACAGCAACGGTGAAGCTTGGTTGTTCGACTTGGACGACGGCATTCTGTATAGCCAGCCGCTTGTGGATAGCTTTCTGTATGCAATCTTTAGTTTGGACGGGAAGCTTATTGCACCAATCTTTATTCCACAAGTGGAGCAAATGCTCAGCGGCCAGCATCTCAGAAACCCTGTCATCGTGGACGGCCGTTTGCATGTGACTACCATAGACGGTGTTGTTGTTGGATTTGACAAGAAGTCGCCAACTCTGGTTGCAGTGAACAAGGCCTGGCAGATATCTCGTCAGAACCGATTAGAAGAGGCCCTCGGAAACCTGCTGTCCAATTGGTCTGATCACAATGATGCGATTGTGTTGCAGGGTGATCCAGAATCAGCGCCGGCATGGTATTATGTTCCATCAAGAGAAATAGTGACCGCCAATGGTCTAACATGGGCGGATCATCCTCTATGGCTAGGAATGGCTGTCGGTGCTGCAACGCCATACATCTACAGCACATCCCGACGCCGTGTGTATGTTGTGGAGAGCAAAAACATGGCCAAAGACAAAGGAGAATTCGCAGCAGCACATTGCTTTGCAAAATCACTTGTGCTACAGAGCATCGTTCCCGAGGAAATAACGATTCCGACGCTGGCTGGCATCGATTTCGCACTCGTTGCAGCGACAAATGCAACCATTGTAATAGACCGAGACAATTGGAAGCATTACAAGGCCATTGTGCTCGACAACAAGGTGGATCAGGACGGTGGCGCTAAGGTGGTAGTCGGCAGTGAGATTCCCATGAAAGTTCTGGTGCGCAAATTTCAGGGAAATCTGGAGTTCTTCGATCCAAACAATGGTAATGTTGTTATACTCCAGAATGTGTTGAAGGGAGACGGAGCTTGTTGCCAGATAGAGATCAAAGTAGGCGATGCTGAGACTATGCTTGTATCCGATTTCTCGGAGATATTCTCAAAGACGCATCCTCTGGTGGCAAGCCAAGTATTTGAGCTTGGGGATGTGATTGCAGTCACAAAACACAAATCGGACTATGCGGATCTGTGGGCCATTCTACGCATGACATAGacgcgtCTTAATTAACTAGTGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGG
example 3: transgenic Cotton line acquisition (see FIG. 3)
The plant over-expression vector VM062-Suit obtained in example 2 was sent to Kyoho Mimi Biotechnology Co., Ltd for transformation, and the transformation background was cotton variety "Bai Cotton No. 1". Transgenic cotton line of T0 generation obtained from the company, T1 generation was cultivated and harvested, SEQ ID NO. 19: with the forward primer: 5'-GACTACAAGGACGACGATGATAAGG-3', respectively; reverse primer: 5'-GTATGCACTCATTTCCTTGAGCTCG-3' is used as primer to carry out PCR amplification to the specific 1400bp segment of the gene of the cyhalothrin, screen T1 positive plants and harvest seeds. The result is shown in figure 4, the target gene is determined not to be subjected to genetic segregation loss, and finally the positive homozygous transgenic cotton containing stable heredity of the cyhalothrin gene is obtained.
Example 4: protein level expression detection of cyhalotoxin gene of transgenic cotton plant
Taking leaves of a transgenic cotton homozygous strain, extracting total protein of the leaves, and using the total protein for Western Blot detection. The method comprises the following specific steps:
1. taking 1-2 g of a fresh sample;
2. the sample was ground in a mortar with liquid nitrogen, placed in a 10mL tube after grinding, and added with a protein extract (0.01M PBS pH 8, 1mM PMSF, 2mM DTT) and shaken at 4 ℃ for 30 min.
3. Centrifuge at 12000rpm for 20min at 4 ℃.
4. And extracting the supernatant, and completing sample preparation.
5. And (3) taking the supernatant, adding a Loading buffer, boiling for 5-10 minutes at 98 ℃, and then determining the protein expression condition by an SDS-polyacrylamide gel electrophoresis method, wherein the figure is 5.
Example 5: insect resistance detection of cycadoxin transgenic cotton plants
The diamondback moth of the second day of the third year of consistent age (average body weight 1.33 mg/head) is taken. Each injection was 2.03. mu.g of periferritin, and the control group was injected with an equal volume (207nL) of Phosphate Buffered Saline (PBS). Three replicates were set and 30 larvae were injected per replicate. Mortality was counted 24h after injection and the results are shown in table 1.
Cotton bollworms aged the second day of the second year (average body weight 2.06 mg/head) were collected at the same age. Each injection was 3.38. mu.g of periferritin, and the control group was injected with an equal volume (345nL) of Phosphate Buffered Saline (PBS). Three replicates were set and 30 larvae were injected per replicate. Mortality was counted 24h after injection and the results are shown in table 1.
TABLE 1 mortality of diamondback moth and Heliothis armigera larvae following cyiron protein injection
Figure BDA0003571196030000741
Two-instar spodoptera frugiperda (average body weight of 1.01 mg/head) with consistent age is taken and used for feeding experiments of spodoptera frugiperda. Before the experiment, the starvation experiment is carried out for 8h in a unified way, then the leaves of the homozygous transgenic cotton strain are taken as an experimental group, and the transgenic receptor material 'Bai Cotton No. 1' and the upland cotton genetic standard system TM-1 are taken as a control group. Three replicates were set and 18 larvae were grown for each replicate. Mortality was counted after 4 days of incubation at 25 ℃ and the results of feeding are shown in FIG. 6 and the mortality statistics are shown in Table 2.
Two-year-old cotton bollworms (average weight of 3.9 mg/head) with consistent age are taken for feeding experiments of the cotton bollworms. Before the experiment, the starvation experiment is carried out for 8h in a unified way, then the leaves of the homozygous transgenic cotton strain are taken as an experimental group, and the transgenic receptor material 'Bai Cotton No. 1' and the upland cotton genetic standard system TM-1 are taken as a control group. Three replicates were set and 18 larvae were grown for each replicate. Mortality was counted after 4 days of incubation at 25 ℃ and the results of feeding are shown in FIG. 6 and the mortality statistics are shown in Table 2.
TABLE 2 Spodoptera frugiperda and Heliothis armigera larvas mortality
Figure BDA0003571196030000751
And II, application embodiment. In order to prove the creativity and the technical value of the technical scheme of the invention, the part is the application example of the technical scheme of the claims on specific products or related technologies.
The invention provides a toxic protein gene from cycas revoluta, which is 6240bp long, 2080 amino acids are coded and introduced into cotton, so that transgenic cotton obtains Spodoptera frugiperda and cotton bollworm resistance, and progeny can be stably inherited. The detailed examples are as follows:
example 1 cloning of a Perferritin Gene.
Example 2.35s construction of plant expression vectors for promoter + cyhalothrin gene.
Example 3. transgenic cotton lines were obtained.
Example 4 protein level expression assay of the ferritin gene of transgenic cotton plants.
Example 5. detection of insect resistance in cyhalothrin transgenic Cotton plants.
And thirdly, evidence of relevant effects of the embodiment. The embodiment of the invention achieves some positive effects in the process of research and development or use, and has great advantages compared with the prior art, and the following contents are described by combining data, diagrams and the like in the test process.
The invention provides a toxin protein gene from cycadrin, which is found for the first time, an insect-resistant gene from cycadrin is introduced into cotton, and the gene is successfully introduced into the cotton and can be stably inherited according to the expression condition of the cycadrin gene (figure 4); according to the expression of the cyiron toxin protein gene (figure 5), the protein coded by the gene is successfully expressed in cotton; according to the insect-resistant experiment results of the transgenic plants (figure 6, table 1 and table 2), the resistance of the transgenic cotton to spodoptera frugiperda reaches 61.11 percent and the resistance to cotton bollworm reaches 59.26 percent. These results, in comparison with other studies, demonstrate that the resistance obtained by the monovalent transgenic cotton of the invention has similar effects to those of the bivalent transgenic cotton studied by others.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of Henan
Shenzhen Huada Institute of Life Sciences
Shenzhen fairy lake botanical garden (Shenzhen garden research center)
<120> insect-resistant cyathrin gene, expression vector and application
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6240
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggttatgg taattgacta tccaagtctc agagcttcgt tatatgccac taagagtgcg 60
gtcccatgtc gcccgcttcc gggcaatcca gagcgggtgt cgcgccgggt atcacgcaga 120
ggcgcgggag agatcgggtc ccatgcagat ttctggatct cccgcgccaa ggcatccgcc 180
atccggattt tcccgcgtca agagggtccg ctccgtgaat cccggagaag attctatctt 240
ctccgcgaag cggtgttcac cggccgcagg accgcctggg ccgctaatcc agcggcccaa 300
cgggcagcat gggaccgcaa gagatcccct ttcttcggaa ggtcttccga tgatattcat 360
tcattcagca agaatccaga agaattcttg actcaaaaca ctctcagctg cagtttatta 420
actcgcaccg ctcagaccgt cacaaaaagc ccagtattgc ccgcccaagg ttgggtcaaa 480
gtgggtcgtg tgccaggaag aggagacgct ctctttgttg agctggttgc cgagaaatca 540
gatgagacag tgaaggcgta cttacttggc tacaatggta agaacgactc ccccgagccg 600
gcatacgttg atattccaaa gaggggcgcc gagaacggaa catttttgtt taccgtggat 660
ctctcgggct gctccgttgt tgtgacacag ttggacgagc agacataccg tgttttccac 720
gacaaacggg tgaacagctc cattctgtac gacaacgtcg taatgtatgt ggatttcaat 780
cactaccgct atcccagagc ccacggacac gggagagata cgggacaagc ggtcgtctgt 840
atgcaattcg ccgacggagt gtggaaattg tttctccagc ggcacgagac ggcgaattct 900
ctcccaaaga tatggtcccg tggcgatggc aaaccggaga ttttcagcga cgccgaaccc 960
cgtccacata tggaagatgg cataaaggaa tttctcaatc ggcgtcgagc tttgcaagac 1020
gaattgaagc acttcgccaa cacgcttgaa gtggacccgg acatagtgtc caacgctgtc 1080
gacggcgaat acgaaggcgg ccagttttcc aggaacgacg atgcaattcg tggatggaac 1140
gaattacgtg caagcatgag acagaaaatg gctgaaagga agaaactgtt gcaaacaaag 1200
aaaggtatgc tagactcatt caacaacccg gatcaccccc tctataaatt tcgcaaacaa 1260
gtgctcgacg agctcaagga aatgagtgca tacgacaaca ccttggaggc atccgagatt 1320
gcggaccgta cttggctgtg gttggagatc aaaaaagcca aaggtacgaa tgcagtggta 1380
aacagtggag cagccccgaa gcagccgagt cttaccgttg gtgaacgcta cgcagagatg 1440
gaggcggccc tttggatgcg tagcgacaca gcggcgtttg agcaagggta tgaatcttat 1500
caaaccgttc gcgtgccggg ttacaatgaa gatatgacat ccattgcaat gaagaagttg 1560
ttcgttgaga gcagtctgac tgacgagcag ctgggcgcct tgtgcaagcg tatatccatt 1620
gctagcgatc aagagaacca tcttctgaca cagaagacgc aggagattga gagcactttc 1680
aaacaatttt cagatggcag ctttttggag tcggcgcctc aggatttctt cctgagacga 1740
gtcggtgata aatctggagg acgctgctat cctcttgtaa gggtaatggc cgtcgcactg 1800
tctgaaggag aagacgttgc ccagaatttt atggagaacc tgaggacatt gtctaggaat 1860
ccagagtatt caggcgaatc ggcaagttca caagccacaa atgcagggtt gttcagagat 1920
gccctggccg gcctgcacgc gaatgtggaa gccattgccg catctgagaa actacctgct 1980
gtgtttggac tgcccgatgt tgctacaatg ttgagccaag cgagcgaaag caaggtttac 2040
gctttgaata ccgaaacaca cagcatgctg gttgcggtga ttgtagacaa acagagtctg 2100
gcgcgcaagt attacttcta tgatcccaac tttctcatgg ccggtttctc cgacataact 2160
cgtttctcgg aagccctcaa gtatcacttt gtgaatctcc agttcgccgg aaactacaaa 2220
gcgtttggca cagcggaggc tccccagttt aacctggtcc ttattgatac gaccgccatg 2280
gaggatgcgc atgttgacgg agggatcacc gtgaaacaat tgagcgaggg cgttgatctt 2340
actaatgtct ttgacagcac caaaaaggct cgtgtggtaa ctgaggcagc agctcgcttg 2400
gctgaagacc aacaactgcg cactggcctc accataatag aggctgacca gtgggcaaac 2460
aggtggcaag atgccgtcat acgattgaaa ctcaaggaag gattaagtgg agagtggatg 2520
ccattgctgg agaaaatgca agaaaacgca gatggttctt atcagattga tttcatcaac 2580
accaagaata ctggggaaat ccgtggggtt tccactagag atgcgacgtt caaagaattc 2640
aaatcctacc tggataaaaa tttagacgta ttgcataagg atttcatctt tcaggatgga 2700
gttttcaaac cactggaggg agagcccatt gacgggttga atgcaatgtt cgcggtccaa 2760
atgattatag attggttcaa ccgggggact aatgctgagg aagatgcggc ctcaggatcc 2820
accaatgccc aactttccct agcgattaaa gtgcacagct acattaacat ggcccaagta 2880
gcgcatactg cagttacaga tgttgccaag gtatacaacc ttgtaaagtc agcactgtat 2940
ggcgagcagg tagctgggga gacatcgctg ttcacatttg ggaaggcgtt gaaatttgca 3000
gcaacagaag ggttgggagt cttactaggg gcgtcctttg tgatcttgga tgcctaccag 3060
ctgtccctcg tggaaaacga tgtgcagaag gccattatcg gcacacaact agcctttgat 3120
tccgcaagct ttctagcggg tattggtgca attgttgctg gagcatcggg cgtgggcatg 3180
attttgggcg gagctggcgt ggtattggga ggcctgggag ttggctttgg agccttagct 3240
caggcattcg gggaaatcac agcagacgcc caactggtgg gcaagtactt cgcactcttg 3300
gacgaggcat accgcggtgg cggatacaga tacgaatcta aagacaaggc attgatgcct 3360
cttgcaggag cagtcatcga caaaatacga ctggccgaca aacatctcca agttcatttc 3420
gacagccaat tcatatatcg aacccatcat ggcagcacag gatccggcaa aattaattac 3480
ttcttctggg ctggcgacat gccgcagatg gtccacgatc gctcccaagc tatcaatgtt 3540
cgagagcaaa ttgggtataa gaacgataat gctactttgg agctaccgga ggatgcctct 3600
gatctgatcc tgcctgccac gcccaagtca tttatcactt accgatacga gattctacca 3660
ttcgctactt cccgccacga tacgggtttc gatgtcatta gacgcctgga agaagactac 3720
agattcgatt acgatttcta catcttccca tctgaataca caatcagaac catcaaacac 3780
gagtacgtag aaacaaaggt ggcagtagtc ctggataggc gctctttgcg actgggagca 3840
cctgagcttc tcaaggagca acatggctat ttgatctatg agatcgaggg gaccggagga 3900
caatacgtta tatgtcttaa ccagggatcc actgtcacac tgaagaccac cggatcagag 3960
gcaaataaaa cattatggat acttgatgcc cgacaactca ttgcaggcaa cgattcaatc 4020
acggtctctc aagatagcat caccatcggc agcacaacag tccatctccg taaccaagat 4080
ttcgattgcc tacagatcgt catgagtcgg gaggagcttc tccaagtgga cttggcgtct 4140
ggagaagccc gcgtgttttc agaagatgcc agtaaatggc tcgacaccaa agaaacaatc 4200
catgaacatc tccataagtt ggctgaagaa cacaggttgc atggccagtt tgtagtggtg 4260
aacaattatt atcgtggagt gggccgtgcc tactatgatg ctgctcacga tcggatgttg 4320
tatacagatg tggaagatga aactctgact cgggatgctg ttcttgccgg cgttacaggc 4380
gaagatgcct acttctacaa tgtaaagcag aatgccctgt ggcgagtgaa tgcaggcaca 4440
caccaagtaa taaccaaata ctatccgacg ttactggagg accctgacag caacatggca 4500
agagttgagc agcatggcaa catcgtcgtg atggccatga gcaggctcaa ccacccggac 4560
aagggagtaa atggaatcat ctatctcgtt catcaagaaa gcatcgtgct tactgctgtg 4620
atcggcaatt cagagatgct caaacgtctt cgccaaacga gtacaattag cttaacagaa 4680
ttgcgaggtg tcaatgacat ggctctggtt tccacgatgg acagattacc cggccaaaag 4740
gttgtcgcta ccaacgctga gtttgtgaca cttctggcta aggatgagca aggagccaat 4800
atcaaggagc agacggtcta ttgccaacaa ggtacgggcc agaccaacga tacatctatt 4860
gaaagcatcc cgattccagg gctctctaat cttttaaccg tgtacggtga agtattcgct 4920
acaactaatg atggtcgcat ccttcgcctg cttggggacg gctcaacatg tctcgagggc 4980
atcaatgagc attggctcat caagcatcca aaatggtggc aggacttggc taccttggag 5040
gcagagaaag ccacgacagt tgcagtattc ggtgttagag acctacaaaa tgcagtggtg 5100
ccaatttggt accacaacgg aaagatcgcg gtggcctcat caaaactgta tggcaaacaa 5160
ctgcaatttt tggggtttaa cagcaacggt gaagcttggt tgttcgactt ggacgacggc 5220
attctgtata gccagccgct tgtggatagc tttctgtatg caatctttag tttggacggg 5280
aagcttattg caccaatctt tattccacaa gtggagcaaa tgctcagcgg ccagcatctc 5340
agaaaccctg tcatcgtgga cggccgtttg catgtgacta ccatagacgg tgttgttgtt 5400
ggatttgaca agaagtcgcc aactctggtt gcagtgaaca aggcctggca gatatctcgt 5460
cagaaccgat tagaagaggc cctcggaaac ctgctgtcca attggtctga tcacaatgat 5520
gcgattgtgt tgcagggtga tccagaatca gcgccggcat ggtattatgt tccatcaaga 5580
gaaatagtga ccgccaatgg tctaacatgg gcggatcatc ctctatggct aggaatggct 5640
gtcggtgctg caacgccata catctacagc acatcccgac gccgtgtgta tgttgtggag 5700
agcaaaaaca tggccaaaga caaaggagaa ttcgcagcag cacattgctt tgcaaaatca 5760
cttgtgctac agagcatcgt tcccgaggaa ataacgattc cgacgctggc tggcatcgat 5820
ttcgcactcg ttgcagcgac aaatgcaacc attgtaatag accgagacaa ttggaagcat 5880
tacaaggcca ttgtgctcga caacaaggtg gatcaggacg gtggcgctaa ggtggtagtc 5940
ggcagtgaga ttcccatgaa agttctggtg cgcaaatttc agggaaatct ggagttcttc 6000
gatccaaaca atggtaatgt tgttatactc cagaatgtgt tgaagggaga cggagcttgt 6060
tgccagatag agatcaaagt aggcgatgct gagactatgc ttgtatccga tttctcggag 6120
atattctcaa agacgcatcc tctggtggca agccaagtat ttgagcttgg ggatgtgatt 6180
gcagtcacaa aacacaaatc ggactatgcg gatctgtggg ccattctacg catgacatag 6240
<210> 2
<211> 5985
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 4950
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 5862
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 6087
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 6240
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggtgatgg ttattgatta cccttctttg agggcttctt tgtatgctac taagtctgct 60
gttccttgta ggcctcttcc tggtaaccct gaaagggttt ctaggagggt ttctagaagg 120
ggtgctggtg aaattggtag tcatgctgat ttttggattt ctagagctaa ggcttctgct 180
attaggattt ttcctagaca agaaggtcca ttgagggaaa gcaggaggag gttctatttg 240
ttgagagagg ctgttttcac aggtaggagg actgcatggg ctgcaaatcc tgctgctcag 300
agagcagctt gggatagaaa gaggtctcct ttcttcggta ggtcatcaga tgatattcat 360
tcattcagta agaacccaga agagttcctt actcaaaata ctttgtcttg ttctcttctt 420
actagaactg ctcaaactgt tactaagtct cctgttttgc ctgctcaagg ttgggttaag 480
gttggtagag tgccaggtag aggtgatgct cttttcgttg agcttgttgc tgaaaaatct 540
gatgaaactg ttaaggctta cttgttgggt tataatggta agaatgattc tcctgaacct 600
gcttatgttg atattccaaa gaggggtgca gagaatggaa ctttcctttt tacagtggat 660
cttagcggtt gttctgttgt tgttactcaa ttggatgagc agacttatag agtgttccat 720
gataagaggg ttaattcaag tattctttac gataacgtgg ttatgtatgt tgattttaac 780
cattacaggt accctagagc tcatggtcat ggtagagata ctggtcaagc tgttgtttgt 840
atgcagtttg ctgatggtgt ttggaagttg ttcttgcaaa gacatgagac tgctaattct 900
cttccaaaga tttggtctag aggtgatggt aagcctgaaa ttttctctga tgctgagcct 960
agacctcata tggaggatgg tattaaggag ttccttaata gaaggagggc tttgcaagat 1020
gaattgaagc attttgctaa tactttggaa gttgatcctg atattgtttc taatgctgtt 1080
gatggtgaat atgaaggtgg tcaattttct agaaatgatg atgcaattag gggttggaat 1140
gaattgagag cttctatgag acagaagatg gctgaaagaa agaagcttct tcaaactaag 1200
aagggtatgt tggatagttt caataatcca gatcatcctc tttacaagtt cagaaaacaa 1260
gttttggatg aattgaaaga gatgtcagct tatgataata cacttgaagc ttctgaaatc 1320
gctgatagga cttggctttg gcttgaaatt aagaaggcta agggaacaaa tgcagttgtt 1380
aatagcggag ctgcacctaa gcaaccatcc cttactgttg gtgaaagata tgctgaaatg 1440
gaagcagcac tttggatgag aagcgataca gctgctttcg aacaaggata tgaaagttat 1500
caaactgtta gagttccagg ttataatgaa gatatgacaa gcattgctat gaagaagttg 1560
tttgttgaat cttccttgac agatgaacaa cttggtgctc tttgtaagag gatttccatt 1620
gctagtgatc aagaaaatca tttgttgact caaaagaccc aagaaatcga atcaactttc 1680
aagcaattct ccgatggaag cttccttgaa tctgctccac aagatttctt ccttagaaga 1740
gttggtgata aatccggtgg taggtgctac ccacttgtta gggttatggc tgttgctctt 1800
tctgaaggtg aagatgttgc tcaaaatttc atggaaaatt tgaggacttt gtcaaggaat 1860
cctgaatatt ccggtgaatc agcttcttct caagctacaa atgctggtct tttcagagat 1920
gctcttgctg gtcttcatgc aaacgttgaa gctatcgctg catccgagaa gcttcctgct 1980
gtttttggtt tgcctgatgt tgctactatg ctttctcagg cttctgaatc taaggtttat 2040
gctcttaata ctgaaactca ttctatgctt gttgctgtta ttgttgataa gcaatctttg 2100
gctaggaagt attattttta cgatcctaat ttccttatgg ctggtttctc tgatattact 2160
agattctccg aagctcttaa gtaccatttc gttaatttgc agtttgcagg taattataag 2220
gctttcggta ctgctgaagc tcctcaattc aatcttgttc ttattgatac taccgctatg 2280
gaggatgctc atgttgatgg tggtattact gtgaagcaac tttctgaagg agttgatctt 2340
actaatgttt tcgattctac taagaaggct agagtggtta ctgaggctgc agctaggttg 2400
gctgaagatc agcaattgag aactggactt acaattattg aagctgatca gtgggctaat 2460
agatggcagg atgctgttat taggttgaag cttaaggaag gtctttctgg tgagtggatg 2520
cctttgcttg aaaagatgca agaaaatgca gatggtagtt atcagattga tttcattaac 2580
actaagaaca ctggagaaat taggggagtt tcaactaggg atgctacttt taaggaattc 2640
aagtcttatt tggataagaa ccttgatgtg ttgcataagg attttatttt ccaggatgga 2700
gtgttcaaac ctcttgaggg tgagcctatt gatggactta atgctatgtt cgctgttcaa 2760
atgattattg attggttcaa taggggtact aacgcagaag aagatgctgc ttctggttct 2820
actaacgctc aattgtcttt ggctattaag gttcattctt atatcaacat ggcacaagtg 2880
gcacatactg ctgttactga tgtggcaaag gtttataatt tggtgaaatc tgctctttac 2940
ggtgaacaag ttgctggtga aacatcattg ttcactttcg gtaaggctct taaatttgct 3000
gctactgaag gtcttggtgt gttgcttggt gcttcttttg tgatccttga tgcttatcag 3060
ctttctcttg ttgagaatga tgttcagaag gcaattattg gtactcaact tgcttttgat 3120
tctgcttctt ttcttgctgg tattggtgct attgttgctg gtgcttctgg tgttggtatg 3180
attttgggtg gtgctggtgt tgttttgggt ggtttgggtg ttggttttgg tgctttggct 3240
caggcttttg gtgagattac tgctgatgct cagttggttg gtaagtattt tgctttgttg 3300
gatgaggctt ataggggtgg tggttatagg tatgagtcta aggataaggc tttgatgcct 3360
ttggctggtg ctgtgattga taagattaga cttgctgata agcatttgca agttcatttt 3420
gattctcaat tcatctacag gactcatcat ggttctactg gttctggtaa gattaattat 3480
ttcttctggg ctggtgatat gcctcaaatg gttcatgata gatctcaagc tattaatgtt 3540
agggagcaga ttggttataa gaatgataat gctactcttg aacttccaga ggatgcttct 3600
gatcttattc ttcctgcaac tcctaagtct ttcattacat atagatacga gatcttgcca 3660
ttcgctactt ctagacatga tactggtttt gatgttatta gaagacttga ggaggattat 3720
agattcgatt atgatttcta catcttccct tctgaatata ccattagaac tattaagcac 3780
gaatacgttg aaaccaaggt tgctgttgtg cttgatagaa gatctttgag acttggtgct 3840
cctgagcttt tgaaggaaca acatggttat cttatttacg agattgaagg tacaggtggt 3900
caatatgtta tttgtttgaa tcagggttct actgtgactc ttaagacaac tggttctgaa 3960
gcaaataaga ctctttggat tttggatgct aggcaactta ttgctggtaa tgatagtatc 4020
actgtttctc aagattctat tactatcggt tctactactg tgcatcttag gaatcaggat 4080
ttcgattgtc ttcagattgt tatgtctaga gaagaacttc ttcaagttga tttggcttct 4140
ggtgaagcta gagttttctc cgaagatgct tctaagtggc ttgatacaaa ggaaactatt 4200
catgaacacc ttcacaagtt ggctgaagaa cataggcttc atggtcaatt cgttgttgtt 4260
aataattact acaggggtgt tggtagagct tattatgatg ctgctcatga tagaatgctt 4320
tatactgatg ttgaggatga aactcttaca agagatgctg ttcttgctgg tgttactggt 4380
gaagatgctt atttttataa cgttaagcag aacgctttgt ggagagttaa cgctggtact 4440
caccaagtta ttactaagta ttatcctact cttcttgagg atcctgattc taacatggct 4500
agagttgagc aacatggtaa tatcgttgtt atggctatgt caagattgaa tcatcctgat 4560
aagggtgtta acggtattat ttatcttgtt catcaggaat ctatcgttct tactgctgtt 4620
attggaaact ctgaaatgct taagagactt agacaaactt ctactatctc tcttactgaa 4680
cttaggggtg ttaacgatat ggctcttgtt tcaactatgg ataggcttcc tggtcagaag 4740
gtggtggcta caaatgcaga attcgttaca cttttggcta aggatgaaca aggagctaat 4800
attaaggaac aaactgttta ttgccagcaa ggtactggtc aaactaatga tacttctatt 4860
gaatcaatcc ctatccctgg tttgtctaat cttcttacag tttatggtga ggttttcgct 4920
acaactaatg atggtagaat ccttagattg cttggtgatg gaagcacttg tcttgagggt 4980
atcaatgaac attggttgat taagcatcct aaatggtggc aggatcttgc tactcttgag 5040
gcagagaagg ctaccactgt tgctgttttt ggagttaggg atttgcaaaa tgctgttgtt 5100
cctatttggt atcataacgg taagattgca gttgcttcat ctaaacttta tggtaagcaa 5160
ttgcagtttt tgggttttaa ttctaacgga gaagcttggc ttttcgatct tgatgatggt 5220
attttgtatt ctcaaccttt ggttgattct ttcctttatg ctattttcag tttggatggt 5280
aagttgattg ctcctatctt tattcctcag gttgaacaaa tgttgtccgg tcaacatctt 5340
aggaaccctg ttattgttga tggtaggctt catgttacta ctattgatgg tgttgttgtt 5400
ggtttcgata agaagtctcc tactcttgtg gctgttaata aggcttggca aatttctaga 5460
caaaataggc ttgaggaggc tttgggtaat ttgttgtcta attggtctga tcataacgat 5520
gctattgttc ttcagggtga tcctgagtct gctcctgctt ggtattatgt tccttctagg 5580
gagattgtga ctgctaatgg tttgacttgg gctgatcacc ctctttggct tggaatggct 5640
gttggtgctg ctactcctta tatttattct acttctagaa ggagggttta tgttgttgag 5700
tctaagaata tggctaagga taagggtgaa tttgctgctg ctcattgttt tgctaagtct 5760
ttggttcttc agtctattgt tcctgaggaa attactattc caactcttgc tggtatcgat 5820
ttcgctttgg ttgctgctac taatgctact attgttattg atagggataa ctggaagcat 5880
tataaggcaa ttgttcttga taacaaggtt gatcaagatg gaggtgctaa ggttgtggtt 5940
ggttctgaga ttcctatgaa ggttcttgtg aggaagtttc aaggtaattt ggaatttttc 6000
gatcctaata acggtaatgt ggttattctt caaaacgttc ttaagggaga tggtgcttgt 6060
tgtcagattg agattaaggt tggtgatgca gagactatgt tggtttctga tttctctgag 6120
attttctcta agactcatcc tttggttgct tctcaggttt tcgaacttgg tgatgttatt 6180
gctgttacta agcataagag tgattatgct gatctttggg ctattttgag gatgacttga 6240
<210> 7
<211> 5985
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
<210> 8
<211> 4590
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 5862
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 6087
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 2079
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Val Met Val Ile Asp Tyr Pro Ser Leu Arg Ala Ser Leu Tyr Ala
1 5 10 15
Thr Lys Ser Ala Val Pro Cys Arg Pro Leu Pro Gly Asn Pro Glu Arg
20 25 30
Val Ser Arg Arg Val Ser Arg Arg Gly Ala Gly Glu Ile Gly Ser His
35 40 45
Ala Asp Phe Trp Ile Ser Arg Ala Lys Ala Ser Ala Ile Arg Ile Phe
50 55 60
Pro Arg Gln Glu Gly Pro Leu Arg Glu Ser Arg Arg Arg Phe Tyr Leu
65 70 75 80
Leu Arg Glu Ala Val Phe Thr Gly Arg Arg Thr Ala Trp Ala Ala Asn
85 90 95
Pro Ala Ala Gln Arg Ala Ala Trp Asp Arg Lys Arg Ser Pro Phe Phe
100 105 110
Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu Glu
115 120 125
Phe Leu Thr Gln Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr Ala
130 135 140
Gln Thr Val Thr Lys Ser Pro Val Leu Pro Ala Gln Gly Trp Val Lys
145 150 155 160
Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Phe Val Glu Leu Val
165 170 175
Ala Glu Lys Ser Asp Glu Thr Val Lys Ala Tyr Leu Leu Gly Tyr Asn
180 185 190
Gly Lys Asn Asp Ser Pro Glu Pro Ala Tyr Val Asp Ile Pro Lys Arg
195 200 205
Gly Ala Glu Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly Cys
210 215 220
Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His
225 230 235 240
Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr
245 250 255
Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg
260 265 270
Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp
275 280 285
Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile
290 295 300
Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu Pro
305 310 315 320
Arg Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg
325 330 335
Ala Leu Gln Asp Glu Leu Lys His Phe Ala Asn Thr Leu Glu Val Asp
340 345 350
Pro Asp Ile Val Ser Asn Ala Val Asp Gly Glu Tyr Glu Gly Gly Gln
355 360 365
Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala
370 375 380
Ser Met Arg Gln Lys Met Ala Glu Arg Lys Lys Leu Leu Gln Thr Lys
385 390 395 400
Lys Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr Lys
405 410 415
Phe Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr Asp
420 425 430
Asn Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp Leu
435 440 445
Glu Ile Lys Lys Ala Lys Gly Thr Asn Ala Val Val Asn Ser Gly Ala
450 455 460
Ala Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu Met
465 470 475 480
Glu Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln Gly
485 490 495
Tyr Glu Ser Tyr Gln Thr Val Arg Val Pro Gly Tyr Asn Glu Asp Met
500 505 510
Thr Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr Asp
515 520 525
Glu Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp Gln
530 535 540
Glu Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr Phe
545 550 555 560
Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe
565 570 575
Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu
580 585 590
Val Arg Val Met Ala Val Ala Leu Ser Glu Gly Glu Asp Val Ala Gln
595 600 605
Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser
610 615 620
Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp
625 630 635 640
Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu
645 650 655
Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser
660 665 670
Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser
675 680 685
Met Leu Val Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr
690 695 700
Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr
705 710 715 720
Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala
725 730 735
Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu
740 745 750
Val Leu Ile Asp Thr Thr Ala Met Glu Asp Ala His Val Asp Gly Gly
755 760 765
Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe
770 775 780
Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu
785 790 795 800
Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp
805 810 815
Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys
820 825 830
Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu
835 840 845
Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr
850 855 860
Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe
865 870 875 880
Lys Ser Tyr Leu Asp Lys Asn Leu Asp Val Leu His Lys Asp Phe Ile
885 890 895
Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly
900 905 910
Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg
915 920 925
Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln
930 935 940
Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val
945 950 955 960
Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys
965 970 975
Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Glu Thr Ser Leu Phe Thr
980 985 990
Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu
995 1000 1005
Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val
1010 1015 1020
Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp
1025 1030 1035 1040
Ser Ala Ser Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser
1045 1050 1055
Gly Val Gly Met Ile Leu Gly Gly Ala Gly Val Val Leu Gly Gly Leu
1060 1065 1070
Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr Ala
1075 1080 1085
Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr
1090 1095 1100
Arg Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro
1105 1110 1115 1120
Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu
1125 1130 1135
Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser
1140 1145 1150
Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met Pro
1155 1160 1165
Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile
1170 1175 1180
Gly Tyr Lys Asn Asp Asn Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser
1185 1190 1195 1200
Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr
1205 1210 1215
Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Thr Gly Phe Asp Val
1220 1225 1230
Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile
1235 1240 1245
Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu
1250 1255 1260
Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala
1265 1270 1275 1280
Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu
1285 1290 1295
Gly Thr Gly Gly Gln Tyr Val Ile Cys Leu Asn Gln Gly Ser Thr Val
1300 1305 1310
Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu
1315 1320 1325
Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln
1330 1335 1340
Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp
1345 1350 1355 1360
Phe Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln Val
1365 1370 1375
Asp Leu Ala Ser Gly Glu Ala Arg Val Phe Ser Glu Asp Ala Ser Lys
1380 1385 1390
Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala
1395 1400 1405
Glu Glu His Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr
1410 1415 1420
Arg Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu
1425 1430 1435 1440
Tyr Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala
1445 1450 1455
Gly Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala
1460 1465 1470
Leu Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr
1475 1480 1485
Pro Thr Leu Leu Glu Asp Pro Asp Ser Asn Met Ala Arg Val Glu Gln
1490 1495 1500
His Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp
1505 1510 1515 1520
Lys Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Ile Val
1525 1530 1535
Leu Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln
1540 1545 1550
Thr Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala
1555 1560 1565
Leu Val Ser Thr Met Asp Arg Leu Pro Gly Gln Lys Val Val Ala Thr
1570 1575 1580
Asn Ala Glu Phe Val Thr Leu Leu Ala Lys Asp Glu Gln Gly Ala Asn
1585 1590 1595 1600
Ile Lys Glu Gln Thr Val Tyr Cys Gln Gln Gly Thr Gly Gln Thr Asn
1605 1610 1615
Asp Thr Ser Ile Glu Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Leu
1620 1625 1630
Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asn Asp Gly Arg Ile Leu
1635 1640 1645
Arg Leu Leu Gly Asp Gly Ser Thr Cys Leu Glu Gly Ile Asn Glu His
1650 1655 1660
Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu
1665 1670 1675 1680
Ala Glu Lys Ala Thr Thr Val Ala Val Phe Gly Val Arg Asp Leu Gln
1685 1690 1695
Asn Ala Val Val Pro Ile Trp Tyr His Asn Gly Lys Ile Ala Val Ala
1700 1705 1710
Ser Ser Lys Leu Tyr Gly Lys Gln Leu Gln Phe Leu Gly Phe Asn Ser
1715 1720 1725
Asn Gly Glu Ala Trp Leu Phe Asp Leu Asp Asp Gly Ile Leu Tyr Ser
1730 1735 1740
Gln Pro Leu Val Asp Ser Phe Leu Tyr Ala Ile Phe Ser Leu Asp Gly
1745 1750 1755 1760
Lys Leu Ile Ala Pro Ile Phe Ile Pro Gln Val Glu Gln Met Leu Ser
1765 1770 1775
Gly Gln His Leu Arg Asn Pro Val Ile Val Asp Gly Arg Leu His Val
1780 1785 1790
Thr Thr Ile Asp Gly Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr
1795 1800 1805
Leu Val Ala Val Asn Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu
1810 1815 1820
Glu Glu Ala Leu Gly Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp
1825 1830 1835 1840
Ala Ile Val Leu Gln Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr
1845 1850 1855
Val Pro Ser Arg Glu Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp
1860 1865 1870
His Pro Leu Trp Leu Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile
1875 1880 1885
Tyr Ser Thr Ser Arg Arg Arg Val Tyr Val Val Glu Ser Lys Asn Met
1890 1895 1900
Ala Lys Asp Lys Gly Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser
1905 1910 1915 1920
Leu Val Leu Gln Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu
1925 1930 1935
Ala Gly Ile Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val
1940 1945 1950
Ile Asp Arg Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn
1955 1960 1965
Lys Val Asp Gln Asp Gly Gly Ala Lys Val Val Val Gly Ser Glu Ile
1970 1975 1980
Pro Met Lys Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe
1985 1990 1995 2000
Asp Pro Asn Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly
2005 2010 2015
Asp Gly Ala Cys Cys Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr
2020 2025 2030
Met Leu Val Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu
2035 2040 2045
Val Ala Ser Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys
2050 2055 2060
His Lys Ser Asp Tyr Ala Asp Leu Trp Ala Ile Leu Arg Met Thr
2065 2070 2075
<210> 12
<211> 1994
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Glu Glu Ala Asp Met Glu Asn Arg Gly Tyr Glu Glu Arg His Ser
1 5 10 15
Gln Ser Gln Ser Ala Thr Pro Leu Arg Arg Arg Lys Arg Ser Pro Phe
20 25 30
Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu
35 40 45
Glu Phe Leu Thr Lys Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr
50 55 60
Ala Gln Thr Val Thr Lys Ser Pro Val Leu Pro Ala Gln Gly Trp Val
65 70 75 80
Lys Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Ser Gly Cys Ser
85 90 95
Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe His Asp
100 105 110
Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met Tyr Val
115 120 125
Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly Arg Asp
130 135 140
Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val Trp Lys
145 150 155 160
Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys Ile Trp
165 170 175
Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu Pro Arg
180 185 190
Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg Arg Ala
195 200 205
Leu Gln Asp Glu Leu Lys His Phe Ala Asn Thr Leu Glu Val Asp Pro
210 215 220
Glu Ile Val Ser Asn Ala Val Asp Gly Glu Tyr Glu Gly Gly Gln Phe
225 230 235 240
Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg Ala Ser
245 250 255
Met Arg Gln Lys Met Ala Glu Arg Lys Lys Arg Leu Glu Arg Asn Lys
260 265 270
Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr Lys Phe
275 280 285
Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr Asp Asn
290 295 300
Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp Leu Glu
305 310 315 320
Ile Lys Lys Ala Lys Gly Thr Asn Ala Val Val Asn Ser Gly Ala Ala
325 330 335
Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu Met Glu
340 345 350
Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln Gly Tyr
355 360 365
Glu Ser Tyr Gln Thr Leu Arg Val Pro Gly Tyr Asn Glu Asp Met Thr
370 375 380
Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr Asp Glu
385 390 395 400
Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp Gln Glu
405 410 415
Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr Phe Lys
420 425 430
Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp Phe Phe
435 440 445
Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro Leu Val
450 455 460
Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn
465 470 475 480
Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly
485 490 495
Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala
500 505 510
Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys
515 520 525
Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln
530 535 540
Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met
545 550 555 560
Leu Val Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr
565 570 575
Phe Tyr Asp Pro Asn Phe Leu Ile Ala Gly Phe Ser Asp Ile Thr Arg
580 585 590
Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly
595 600 605
Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val
610 615 620
Leu Ile Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly Gly Ile
625 630 635 640
Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp
645 650 655
Ser Thr Lys Lys Ala Arg Val Val Thr Glu Thr Ala Ala Arg Leu Ala
660 665 670
Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln
675 680 685
Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu
690 695 700
Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln Glu Asn
705 710 715 720
Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn Thr Gly
725 730 735
Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys
740 745 750
Ser Tyr Leu Asp Lys Asn Leu Asp Val Leu His Lys Asp Phe Ile Phe
755 760 765
Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp Gly Leu
770 775 780
Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Arg Gly
785 790 795 800
Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu
805 810 815
Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala
820 825 830
His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys Ser
835 840 845
Ala Leu Tyr Gly Glu Gln Val Ala Gly Glu Ala Ser Leu Leu Thr Phe
850 855 860
Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu
865 870 875 880
Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu
885 890 895
Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser
900 905 910
Ala Ser Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser Gly
915 920 925
Val Gly Met Ile Leu Gly Gly Ala Gly Val Val Leu Gly Gly Leu Gly
930 935 940
Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr Ala Asp
945 950 955 960
Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Arg
965 970 975
Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu
980 985 990
Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His Leu Gln
995 1000 1005
Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Leu Thr
1010 1015 1020
Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met Pro Gln
1025 1030 1035 1040
Met Val His Asp Arg Ser Gln Ala Ile Asn Ile Arg Glu Gln Ile Gly
1045 1050 1055
Tyr Lys Asn Asp His Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp
1060 1065 1070
Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Gln Tyr Gln
1075 1080 1085
Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile
1090 1095 1100
Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe
1105 1110 1115 1120
Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr
1125 1130 1135
Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro
1140 1145 1150
Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly
1155 1160 1165
Thr Gly Gly Gln Tyr Val Ile Cys Leu Asn Gln Gly Ser Thr Val Thr
1170 1175 1180
Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp
1185 1190 1195 1200
Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp
1205 1210 1215
Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe
1220 1225 1230
Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln Val Asp
1235 1240 1245
Leu Ala Ser Gly Glu Ala Arg Val Phe Ser Glu Asp Ala Ser Lys Trp
1250 1255 1260
Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Glu
1265 1270 1275 1280
Glu His Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr Arg
1285 1290 1295
Gly Val Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr
1300 1305 1310
Thr Asp Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly
1315 1320 1325
Val Thr Gly Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu
1330 1335 1340
Trp Arg Val Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro
1345 1350 1355 1360
Thr Leu Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His
1365 1370 1375
Gly Asn Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp Lys
1380 1385 1390
Gly Val Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu
1395 1400 1405
Thr Ala Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr
1410 1415 1420
Ser Thr Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu
1425 1430 1435 1440
Val Ser Thr Met Asp Lys Leu Pro Gly Gln Lys Val Val Ala Thr Asn
1445 1450 1455
Ala Glu Phe Val Thr Ile Leu Ala Lys Asp Glu Gln Gly Ser Gln Val
1460 1465 1470
Cys Phe Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile
1475 1480 1485
Gln Pro Pro Ile Pro Thr Asp Leu Thr Leu Thr Val Ser Ser His Lys
1490 1495 1500
Val Ala Gly Gln Asp Val Phe Tyr Phe Tyr Ser Ile Lys Glu Gln Thr
1505 1510 1515 1520
Val Tyr Cys Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Ile Glu
1525 1530 1535
Ser Ile Pro Ile Pro Gly Leu Ser Asn Leu Leu Thr Val Tyr Gly Glu
1540 1545 1550
Val Phe Ala Thr Thr Asp Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp
1555 1560 1565
Gly Ser Thr Tyr Leu Asp Gly Ile Asn Glu His Trp Leu Ile Lys His
1570 1575 1580
Pro Lys Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr
1585 1590 1595 1600
Thr Val Ala Val Phe Gly Val Arg Asp Leu Gln Asn Ala Val Val Pro
1605 1610 1615
Ile Trp Tyr His Asn Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr
1620 1625 1630
Gly Lys Gln Leu Gln Phe Leu Gly Phe Asn Ser Asn Gly Glu Ala Trp
1635 1640 1645
Leu Phe Asp Leu Asp Asp Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp
1650 1655 1660
Ser Phe Leu Tyr Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro
1665 1670 1675 1680
Ile Phe Ile Pro Gln Val Glu Gln Met Leu Ser Gly Gln His Leu Arg
1685 1690 1695
Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly
1700 1705 1710
Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn
1715 1720 1725
Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly
1730 1735 1740
Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln
1745 1750 1755 1760
Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu
1765 1770 1775
Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu
1780 1785 1790
Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Ser Thr Ser Arg
1795 1800 1805
Arg Arg Val Tyr Ala Val Glu Ser Lys His Met Ala Lys Asp Lys Gly
1810 1815 1820
Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser
1825 1830 1835 1840
Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Ile Asp Phe
1845 1850 1855
Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile His Arg Asp Asn
1860 1865 1870
Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp
1875 1880 1885
Gly Gly Ala Lys Val Val Val Gly Ser Gly Ile Pro Met Lys Val Leu
1890 1895 1900
Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly
1905 1910 1915 1920
Asn Val Val Ile Leu Arg Asn Val Leu Lys Gly Asp Gly Ala Cys Ser
1925 1930 1935
Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp
1940 1945 1950
Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val
1955 1960 1965
Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr
1970 1975 1980
Ala Asp Leu Trp Ala Ile Leu Arg Met Thr
1985 1990
<210> 13
<211> 1529
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala Gln Asn Phe Met
1 5 10 15
Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr Ser Gly Glu Ser
20 25 30
Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg Asp Ala Leu Ala
35 40 45
Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser Glu Lys Leu Pro
50 55 60
Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu Ser Gln Ala Ser
65 70 75 80
Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His Ser Met Leu Ala
85 90 95
Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys Tyr Tyr Phe Tyr
100 105 110
Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile Thr Arg Phe Ser
115 120 125
Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe Ala Gly Asn Tyr
130 135 140
Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn Leu Val Leu Ile
145 150 155 160
Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly Gly Ile Thr Val
165 170 175
Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val Phe Asp Ser Thr
180 185 190
Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg Leu Ala Glu Asp
195 200 205
Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala Asp Gln Trp Ala
210 215 220
Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu Lys Glu Gly Leu
225 230 235 240
Ser Gly Glu Trp Met Pro Leu Leu Glu Arg Met Gln Glu Asn Gly His
245 250 255
Gly Ser Tyr Gln Ile Glu Phe Leu Asn Thr Lys Asn Thr Gly Glu Thr
260 265 270
Arg Trp Val Ser Thr Arg Asp Ala Thr Phe Lys Glu Phe Lys Ser Tyr
275 280 285
Leu Asp Lys Asn Phe Asp Ala Leu Arg Lys Asp Phe Ile Phe Gln Asp
290 295 300
Gly Val Phe Lys Pro Leu Asp Gly Glu Pro Ile Asp Gly Leu Asn Ala
305 310 315 320
Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn Pro Gly Thr Asn
325 330 335
Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala Gln Leu Ser Leu
340 345 350
Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln Val Ala His Thr
355 360 365
Ala Val Thr Asp Val Ala Lys Val Tyr Asn Leu Val Lys Ser Ala Leu
370 375 380
Tyr Gly Glu Gln Val Ala Gly Glu Ala Ser Leu Leu Thr Phe Gly Lys
385 390 395 400
Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val Leu Leu Gly Ala
405 410 415
Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu Val Glu Asn Asp
420 425 430
Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe Asp Ser Ala Ser
435 440 445
Phe Leu Ala Gly Ile Gly Ala Ile Val Ala Gly Ala Ser Gly Val Gly
450 455 460
Met Ile Leu Gly Gly Ala Gly Val Ile Leu Gly Gly Leu Gly Val Gly
465 470 475 480
Phe Gly Ala Leu Ala Gln Ala Phe Gly Gln Ile Thr Ala Asp Ala Gln
485 490 495
Leu Val Gly Glu Tyr Phe Ala Leu Leu Asp Glu Ala Tyr Ser Gly Gly
500 505 510
Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met Pro Leu Ala Gly
515 520 525
Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys Arg Leu Gln Val His
530 535 540
Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly Ser Thr Gly Ser
545 550 555 560
Gly Lys Ile Asn Tyr Phe Phe Trp Val Gly Asp Met Pro Gln Met Val
565 570 575
His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln Ile Gly Tyr Lys
580 585 590
Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala Ser Asp Leu Ile
595 600 605
Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg Tyr Glu Ile Leu
610 615 620
Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp Val Ile Arg Arg
625 630 635 640
Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr Ile Phe Pro Ser
645 650 655
Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val Glu Thr Lys Val
660 665 670
Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly Ala Pro Glu Leu
675 680 685
Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile Glu Gly Thr Gly
690 695 700
Gly Gln Tyr Val Ile Cys Leu Asn His Gly Ser Thr Val Thr Leu Lys
705 710 715 720
Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile Leu Asp Ala Arg
725 730 735
Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser Gln Asp Ser Ile
740 745 750
Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln Asp Phe Asp Cys
755 760 765
Leu Gln Ile Val Met Ser Arg Asp Glu Leu Leu Gln Val Asp Leu Ala
770 775 780
Ser Arg Glu Val Arg Val Phe Ser Glu Asp Ala Ser Lys Trp Leu Asp
785 790 795 800
Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu Ala Gly Glu His
805 810 815
Arg Leu His Gly Gln Phe Val Val Val Asn Asn Tyr Tyr Arg Gly Val
820 825 830
Gly Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp
835 840 845
Val Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr
850 855 860
Gly Glu Asp Ala Tyr Phe Tyr Asn Val Asn Gln Asn Ala Leu Trp Arg
865 870 875 880
Val Asn Ala Asp Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Leu
885 890 895
Leu Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His Gly Asn
900 905 910
Ile Val Val Met Ala Met Ser Arg Leu Asn His Pro Asp Lys Gly Val
915 920 925
Asn Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala
930 935 940
Val Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr
945 950 955 960
Ile Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser
965 970 975
Thr Met Asp Arg Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu
980 985 990
Phe Val Thr Leu Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe
995 1000 1005
Trp Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro
1010 1015 1020
Pro Ile Pro Thr Asp Leu Thr Leu Thr Val Ser Ser His Lys Val Ala
1025 1030 1035 1040
Gly Gln Asp Val Phe Tyr Phe Tyr Ser Ile Lys Glu Gln Thr Val Tyr
1045 1050 1055
Cys Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Ile Glu Ser Ile
1060 1065 1070
Pro Ile Pro Gly Leu Ser Asn Leu Leu Thr Val Tyr Gly Glu Val Phe
1075 1080 1085
Ala Thr Thr Asn Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser
1090 1095 1100
Thr Tyr Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys
1105 1110 1115 1120
Trp Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Lys Ala Thr Thr Val
1125 1130 1135
Ala Val Phe Gly Val Arg Asp Pro Gln Asn Ala Val Val Pro Ile Trp
1140 1145 1150
Tyr His Asn Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys
1155 1160 1165
Gln Leu Gln Phe Leu Gly Phe Asn Ser Asn Gly Glu Ala Trp Leu Phe
1170 1175 1180
Asp Leu Asp Asp Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser Gln
1185 1190 1195 1200
Ala Val Asp Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Asn
1205 1210 1215
Leu Ile Pro Gln Val Glu Gln Met Leu Ser Gly Gln His Leu Arg Asn
1220 1225 1230
Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val
1235 1240 1245
Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys
1250 1255 1260
Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn
1265 1270 1275 1280
Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly
1285 1290 1295
Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile
1300 1305 1310
Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly
1315 1320 1325
Met Ala Val Gly Ala Ala Thr Leu Tyr Ile Tyr Ser Thr Ser Arg Arg
1330 1335 1340
Arg Val Tyr Ala Val Glu Ser Lys Lys Met Ala Lys Asp Lys Gly Glu
1345 1350 1355 1360
Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser Ile
1365 1370 1375
Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Thr Asp Phe Ala
1380 1385 1390
Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile Asp Arg Asp Asn Trp
1395 1400 1405
Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp Gly
1410 1415 1420
Gly Ala Lys Val Val Val Gly Ser Glu Ile Pro Met Lys Val Leu Val
1425 1430 1435 1440
Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly Asn
1445 1450 1455
Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Ser Lys
1460 1465 1470
Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp Phe
1475 1480 1485
Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val Phe
1490 1495 1500
Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser Asp Tyr Ala
1505 1510 1515 1520
Asp Leu Trp Ala Ile Leu Arg Met Thr
1525
<210> 14
<211> 1953
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Glu Glu Val Asp Met Gln Asn Arg Gly Tyr Glu Glu Trp His Ser
1 5 10 15
Gln Ser Gln Ser Ala Thr Thr Leu Arg Arg Arg Lys Arg Ser Pro Val
20 25 30
Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu
35 40 45
Glu Phe Leu Thr Gln Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr
50 55 60
Ala Glu Thr Val Thr Lys Ser Pro Val Leu Pro Glu Arg Gly Trp Val
65 70 75 80
Lys Val Gly Arg Val Pro Gly Arg Gly Asp Ala Leu Phe Val Gln Val
85 90 95
Val Ala Glu Gln Ser Tyr Asp Thr Val Lys Ala Tyr Leu Leu Gly Tyr
100 105 110
Asn Gly Glu Asn Asp Ser Pro Asn Pro Ala Tyr Val Asp Ile Pro Lys
115 120 125
Arg Ala Ala Glu Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly
130 135 140
Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe
145 150 155 160
His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met
165 170 175
Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly
180 185 190
Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val
195 200 205
Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys
210 215 220
Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Glu
225 230 235 240
Pro Arg Gln His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg
245 250 255
Arg Ala Leu Gln Asp Glu Leu Lys Lys Phe Ala Asp Met Phe Gly Val
260 265 270
Asp Pro Glu Ile Val Ser Asn Ala Val Asp Gly Lys Tyr Lys Gly Gly
275 280 285
Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg
290 295 300
Ala Ser Met Arg Gln Lys Met Ala Glu Arg Asn Lys Val Leu Leu Thr
305 310 315 320
Met Lys Leu Glu Gln Pro Arg Ser Ser Arg Val Leu Pro Leu Val Asn
325 330 335
Ala Thr Gln Arg Trp Arg Arg Pro Phe Gly Cys Val Ala Thr Gln Arg
340 345 350
Arg Leu Ser Lys Ala Gly Arg Leu Val Gln Ala Tyr Ile His Arg Asp
355 360 365
Gln Glu Asn His Leu Leu Thr Gln Lys Thr Gln Glu Ile Glu Ser Thr
370 375 380
Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp
385 390 395 400
Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro
405 410 415
Leu Val Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala
420 425 430
Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Lys Asn Pro Glu Tyr
435 440 445
Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg
450 455 460
Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser
465 470 475 480
Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu
485 490 495
Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His
500 505 510
Ser Met Leu Ala Ala Val Ile Val Asp Lys Gln Ser Leu Ala Arg Lys
515 520 525
Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile
530 535 540
Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe
545 550 555 560
Ala Gly Asn Tyr Lys Pro Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn
565 570 575
Leu Val Leu Ile Asp Thr Thr Val Met Ala Asp Ala His Val Asp Gly
580 585 590
Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val
595 600 605
Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg
610 615 620
Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala
625 630 635 640
Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Lys Leu
645 650 655
Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln
660 665 670
Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn
675 680 685
Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Gly Thr Phe Lys Glu
690 695 700
Leu Lys Ser Tyr Leu Asp Lys Asn Leu Asp Ala Leu His Lys Asp Phe
705 710 715 720
Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp
725 730 735
Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn
740 745 750
Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala
755 760 765
Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln
770 775 780
Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Ser Leu Val
785 790 795 800
Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Gly Ala Ser Leu Leu
805 810 815
Thr Phe Gly Lys Ala Leu Lys Leu Ala Ala Thr Glu Gly Leu Gly Val
820 825 830
Leu Leu Gly Ala Pro Val Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu
835 840 845
Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Asp Phe
850 855 860
Asp Ser Ala Ser Phe Leu Val Ser Ile Gly Ala Ala Val Ala Gly Val
865 870 875 880
Ser Gly Val Gly Ile Val Leu Ser Gly Ala Gly Val Leu Leu Ala Gly
885 890 895
Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Glu Ile Thr
900 905 910
Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala
915 920 925
Tyr Arg Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met
930 935 940
Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His
945 950 955 960
Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly
965 970 975
Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Val Gly Asp Met
980 985 990
Pro Lys Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln
995 1000 1005
Ile Gly Tyr Lys Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala
1010 1015 1020
Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg
1025 1030 1035 1040
Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp
1045 1050 1055
Val Ile Arg Arg Leu Glu Glu Asp His Arg Phe Asp Tyr Val Phe Tyr
1060 1065 1070
Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val
1075 1080 1085
Glu Thr Lys Val Ala Val Val Pro Asp Arg Arg Pro Leu Arg Leu Arg
1090 1095 1100
Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile
1105 1110 1115 1120
Glu Gly Thr Gly Gly Glu Tyr Val Ile Cys Leu Asn Lys Gly Ser Thr
1125 1130 1135
Val Thr Leu Phe Thr Thr Gly Ser Glu Ala Asn Lys Thr Ser Trp Ile
1140 1145 1150
Leu Asp Ala Arg Gln Ile Ile Ala Gly Asn Asp Pro Ile Ile Val Ser
1155 1160 1165
Gln Asp Ser Ile Thr Met Gly Ser Thr Thr Val His Leu Arg Asn His
1170 1175 1180
Asp Phe Asp Cys Leu Arg Ile Val Thr Ser Arg Glu Glu Leu Leu Gln
1185 1190 1195 1200
Val Asp Ser Ala Ser Arg Gln Thr Arg Val Leu Ser Glu Asp Ala Ser
1205 1210 1215
Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu
1220 1225 1230
Ala Glu Glu His Arg Leu Leu Gly Gln Phe Val Val Val Asn Asn Tyr
1235 1240 1245
Ser Val Gln Glu Gln Ser Asp Ile Gly Thr Leu Tyr Arg Gly Val Gly
1250 1255 1260
Arg Ala His Asp Arg Met Leu Tyr Thr Asp Val Glu Asp Glu Thr Leu
1265 1270 1275 1280
Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly Glu Asp Ala Tyr Phe
1285 1290 1295
Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val Asn Ala Gly Thr His
1300 1305 1310
Gln Val Ile Thr Lys Tyr Tyr Pro Thr Val Leu Glu Asp Pro Asp Ser
1315 1320 1325
Lys Met Ala Arg Val Glu Gln Arg Gly Asn Ile Val Val Met Ala Met
1330 1335 1340
Ser Arg Leu Asn His Ser Asp Lys Gly Val Asn Gly Ile Ile Tyr Leu
1345 1350 1355 1360
Val His Gln Glu Ser Met Val Leu Thr Ala Val Ile Gly Asn Ser Glu
1365 1370 1375
Met Leu Lys Arg Leu Arg Gln Ala Ser Thr Ile Ser Leu Thr Glu Leu
1380 1385 1390
Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr Met Asp Lys Leu Pro
1395 1400 1405
Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe Val Thr Ile Leu Ala
1410 1415 1420
Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp Leu Arg Thr Val Asp
1425 1430 1435 1440
Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro Ile Pro Thr Asp Leu
1445 1450 1455
Thr Leu Thr Val Ser Ser His Lys Val Ala Gly Gln Asp Val Phe Tyr
1460 1465 1470
Phe Tyr Ser Ile Lys Glu Gln Arg Val Tyr Arg Gln Gln Gly Thr Gly
1475 1480 1485
Gln Thr Asn Asp Thr Ser Val Gly Ser Ile Pro Ile Pro Gly Leu Ser
1490 1495 1500
Asn Leu Phe Thr Val Tyr Gly Glu Val Phe Ala Thr Thr Asn Asp Gly
1505 1510 1515 1520
Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr Tyr Leu Glu Gly Ile
1525 1530 1535
Asn Glu His Trp Leu Ile Lys His Pro Lys Trp Trp Gln Asp Leu Ala
1540 1545 1550
Thr Leu Glu Ala Glu Lys Ala Thr Thr Ile Ala Val Phe Gly Val Arg
1555 1560 1565
Asp Pro Gln Asn Ala Val Val Pro Ile Cys Asn Gly Glu Ala Trp Leu
1570 1575 1580
Phe Asp Leu Asp Gly Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Ser
1585 1590 1595 1600
Gln Ala Val Asp Ala Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro
1605 1610 1615
Asn Leu Ile Pro Gln Val Gln Gln Met Leu Ser Gly Gln His Leu Arg
1620 1625 1630
Asn Pro Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly
1635 1640 1645
Val Val Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Gly Asn
1650 1655 1660
Lys Ala Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Arg
1665 1670 1675 1680
Asn Leu Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln
1685 1690 1695
Gly Asp Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu
1700 1705 1710
Ile Val Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu
1715 1720 1725
Gly Met Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Ser Thr Ser Arg
1730 1735 1740
Cys Arg Val Tyr Ala Val Glu Ser Lys Asn Met Ala Lys Asp Lys Gly
1745 1750 1755 1760
Glu Phe Ala Ala Ala His Cys Phe Ala Lys Ser Leu Val Leu Gln Ser
1765 1770 1775
Ile Val Pro Ala Glu Ile Thr Ile Pro Met Leu Ala Gly Ile Asp Phe
1780 1785 1790
Ala Leu Val Ala Ala Thr Lys Ala Thr Ile Val Ile Asp Arg Asp Asn
1795 1800 1805
Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp Gln Asp
1810 1815 1820
Gly Gly Ala Lys Val Val Val Gly Ser Glu Ile Pro Met Lys Val Leu
1825 1830 1835 1840
Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn Asn Gly
1845 1850 1855
Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala Cys Ser
1860 1865 1870
Gln Ile Glu Ile Lys Val Gly Asp Ala Glu Thr Met Leu Val Ser Asp
1875 1880 1885
Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser Gln Val
1890 1895 1900
Phe Glu Leu Gly Asp Gly Cys Asp Cys Ser His Lys Thr Gln Ile Gly
1905 1910 1915 1920
Leu Cys Gly Ser Val Gly His Ser Thr His Asp Ile Ala Tyr Ile Ser
1925 1930 1935
His Ala Tyr Leu His Arg Trp Pro Ile Tyr Val Ile Ser Thr Leu Ile
1940 1945 1950
Ala
<210> 15
<211> 2028
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Glu Glu Ala Asp Met Glu Asn Arg Gly Asn Glu Glu Arg His Ser
1 5 10 15
Gln Ser Gln Ser Ala Thr Pro Leu Arg Arg Arg Lys Arg Ser Pro Val
20 25 30
Phe Gly Arg Ser Ser Asp Asp Ile His Ser Phe Ser Lys Asn Pro Glu
35 40 45
Glu Phe Leu Thr Arg Asn Thr Leu Ser Cys Ser Leu Leu Thr Arg Thr
50 55 60
Ala Glu Ala Val Thr Lys Ser Pro Ala Leu Pro Ala Gln Gly Trp Val
65 70 75 80
Lys Val Gly Arg Val Pro Gly Arg Gly Asp Val Leu Phe Val Gln Leu
85 90 95
Val Ala Glu Lys Ser Asp Glu Thr Val Lys Ala Tyr Leu Leu Gly Tyr
100 105 110
Asn Gly Glu Asn Asp Phe Pro Lys Pro Ala Tyr Val Asp Ile Pro Lys
115 120 125
Arg Gly Ala Lys Asn Gly Thr Phe Leu Phe Thr Val Asp Leu Ser Gly
130 135 140
Cys Ser Val Val Val Thr Gln Leu Asp Glu Gln Thr Tyr Arg Val Phe
145 150 155 160
His Asp Lys Arg Val Asn Ser Ser Ile Leu Tyr Asp Asn Val Val Met
165 170 175
Tyr Val Asp Phe Asn His Tyr Arg Tyr Pro Arg Ala His Gly His Gly
180 185 190
Arg Asp Thr Gly Gln Ala Val Val Cys Met Gln Phe Ala Asp Gly Val
195 200 205
Trp Lys Leu Phe Leu Gln Arg His Glu Thr Ala Asn Ser Leu Pro Lys
210 215 220
Ile Trp Ser Arg Gly Asp Gly Lys Pro Glu Ile Phe Ser Asp Ala Asp
225 230 235 240
Pro Arg Pro His Met Glu Asp Gly Ile Lys Glu Phe Leu Asn Arg Arg
245 250 255
Arg Ala Leu Gln Asp Glu Leu Lys Lys Phe Ala Asp Met Phe Gly Val
260 265 270
Asp Pro Glu Ile Val Ser Asn Ala Val Asp Gly Lys Tyr Glu Gly Gly
275 280 285
Gln Phe Ser Arg Asn Asp Asp Ala Ile Arg Gly Trp Asn Glu Leu Arg
290 295 300
Ala Arg Met Arg Gln Lys Met Ala Glu Arg Lys Lys Leu Leu Glu Thr
305 310 315 320
Lys Lys Gly Met Leu Asp Ser Phe Asn Asn Pro Asp His Pro Leu Tyr
325 330 335
Glu Phe Arg Lys Gln Val Leu Asp Glu Leu Lys Glu Met Ser Ala Tyr
340 345 350
Asp Asn Thr Leu Glu Ala Ser Glu Ile Ala Asp Arg Thr Trp Leu Trp
355 360 365
Leu Glu Ile Lys Lys Ala Arg Gly Thr Asn Ala Val Val Asn Ser Gly
370 375 380
Ser Ala Pro Lys Gln Pro Ser Leu Thr Val Gly Glu Arg Tyr Ala Glu
385 390 395 400
Met Glu Ala Ala Leu Trp Met Arg Ser Asp Thr Ala Ala Phe Glu Gln
405 410 415
Gly Tyr Glu Ser Tyr Gln Thr Val Arg Val Pro Gly Tyr Asn Glu Asp
420 425 430
Met Thr Ser Ile Ala Met Lys Lys Leu Phe Val Glu Ser Ser Leu Thr
435 440 445
Asp Glu Gln Leu Gly Ala Leu Cys Lys Arg Ile Ser Ile Ala Ser Asp
450 455 460
Gln Glu Asn His Leu Leu Arg Gln Lys Thr Gln Glu Ile Glu Asn Thr
465 470 475 480
Phe Lys Gln Phe Ser Asp Gly Ser Phe Leu Glu Ser Ala Pro Gln Asp
485 490 495
Phe Phe Leu Arg Arg Val Gly Asp Lys Ser Gly Gly Arg Cys Tyr Pro
500 505 510
Leu Val Arg Val Met Ala Val Ala Leu Ser Gln Gly Glu Asp Val Ala
515 520 525
Gln Asn Phe Met Glu Asn Leu Arg Thr Leu Ser Arg Asn Pro Glu Tyr
530 535 540
Ser Gly Glu Ser Ala Ser Ser Gln Ala Thr Asn Ala Gly Leu Phe Arg
545 550 555 560
Asp Ala Leu Ala Gly Leu His Ala Asn Val Glu Ala Ile Ala Ala Ser
565 570 575
Glu Lys Leu Pro Ala Val Phe Gly Leu Pro Asp Val Ala Thr Met Leu
580 585 590
Ser Gln Ala Ser Glu Ser Lys Val Tyr Ala Leu Asn Thr Glu Thr His
595 600 605
Ser Met Leu Val Ala Val Ile Ala Asp Glu Gln Ser Leu Ala Pro Lys
610 615 620
Tyr Tyr Phe Tyr Asp Pro Asn Phe Leu Met Ala Gly Phe Ser Asp Ile
625 630 635 640
Thr Arg Phe Ser Glu Ala Leu Lys Tyr His Phe Val Asn Leu Gln Phe
645 650 655
Ala Gly Asn Tyr Lys Ala Phe Gly Thr Ala Glu Ala Pro Gln Phe Asn
660 665 670
Leu Val Leu Ile Asp Thr Thr Ala Met Ala Asp Ala His Val Asp Gly
675 680 685
Gly Ile Thr Val Lys Gln Leu Ser Glu Gly Val Asp Leu Thr Asn Val
690 695 700
Phe Asp Ser Thr Lys Lys Ala Arg Val Val Thr Glu Ala Ala Ala Arg
705 710 715 720
Leu Ala Glu Asp Gln Gln Leu Arg Thr Gly Leu Thr Ile Ile Glu Ala
725 730 735
Asp Gln Trp Ala Asn Arg Trp Gln Asp Ala Val Ile Arg Leu Arg Leu
740 745 750
Lys Glu Gly Leu Ser Gly Glu Trp Met Pro Leu Leu Glu Lys Met Gln
755 760 765
Glu Asn Ala Asp Gly Ser Tyr Gln Ile Asp Phe Ile Asn Thr Lys Asn
770 775 780
Thr Gly Glu Ile Arg Gly Val Ser Thr Arg Asp Ala Thr Phe Lys Glu
785 790 795 800
Phe Lys Ser Tyr Leu Asp Lys Asn Leu Asp Ala Leu His Lys Asp Phe
805 810 815
Ile Phe Gln Asp Gly Val Phe Lys Pro Leu Glu Gly Glu Pro Ile Asp
820 825 830
Gly Leu Asn Ala Met Phe Ala Val Gln Met Ile Ile Asp Trp Phe Asn
835 840 845
Arg Gly Thr Asn Ala Glu Glu Asp Ala Ala Ser Gly Ser Thr Asn Ala
850 855 860
Gln Leu Ser Leu Ala Ile Lys Val His Ser Tyr Ile Asn Met Ala Gln
865 870 875 880
Val Ala His Thr Ala Val Thr Asp Val Ala Lys Val Tyr Ser Leu Val
885 890 895
Lys Ser Ala Leu Tyr Gly Glu Gln Val Ala Gly Gly Ala Ser Leu Leu
900 905 910
Thr Phe Gly Lys Ala Leu Lys Phe Ala Ala Thr Glu Gly Leu Gly Val
915 920 925
Leu Leu Gly Ala Ser Phe Val Ile Leu Asp Ala Tyr Gln Leu Ser Leu
930 935 940
Val Glu Asn Asp Val Gln Lys Ala Ile Ile Gly Thr Gln Leu Ala Phe
945 950 955 960
Asp Ser Ala Ser Phe Leu Val Ser Ile Gly Ala Ala Val Ala Gly Val
965 970 975
Ser Gly Val Gly Ile Val Leu Ser Gly Ala Gly Val Leu Leu Ala Gly
980 985 990
Leu Gly Val Gly Phe Gly Ala Leu Ala Gln Ala Phe Gly Gln Ile Thr
995 1000 1005
Ala Asp Ala Gln Leu Val Gly Lys Tyr Phe Ala Leu Leu Asp Glu Ala
1010 1015 1020
Tyr Ser Gly Gly Gly Tyr Arg Tyr Glu Ser Lys Asp Lys Ala Leu Met
1025 1030 1035 1040
Pro Leu Ala Gly Ala Val Ile Asp Lys Ile Arg Leu Ala Asp Lys His
1045 1050 1055
Leu Gln Val His Phe Asp Ser Gln Phe Ile Tyr Arg Thr His His Gly
1060 1065 1070
Ser Thr Gly Ser Gly Lys Ile Asn Tyr Phe Phe Trp Ala Gly Asp Met
1075 1080 1085
Pro Gln Met Val His Asp Arg Ser Gln Ala Ile Asn Val Arg Glu Gln
1090 1095 1100
Ile Gly Tyr Lys Asn Asp Ser Ala Thr Leu Glu Leu Pro Glu Asp Ala
1105 1110 1115 1120
Ser Asp Leu Ile Leu Pro Ala Thr Pro Lys Ser Phe Ile Thr Tyr Arg
1125 1130 1135
Tyr Glu Ile Leu Pro Phe Ala Thr Ser Arg His Asp Arg Gly Phe Asp
1140 1145 1150
Val Ile Arg Arg Leu Glu Glu Asp Tyr Arg Phe Asp Tyr Asp Phe Tyr
1155 1160 1165
Ile Phe Pro Ser Glu Tyr Thr Ile Arg Thr Ile Lys His Glu Tyr Val
1170 1175 1180
Glu Thr Lys Val Ala Val Val Leu Asp Arg Arg Ser Leu Arg Leu Gly
1185 1190 1195 1200
Ala Pro Glu Leu Leu Lys Glu Gln His Gly Tyr Leu Ile Tyr Glu Ile
1205 1210 1215
Glu Gly Thr Gly Gly Gln Tyr Val Val Cys Leu Asn His Gly Ser Thr
1220 1225 1230
Val Thr Leu Lys Thr Thr Gly Ser Glu Ala Asn Lys Thr Leu Trp Ile
1235 1240 1245
Leu Asp Ala Arg Gln Leu Ile Ala Gly Asn Asp Ser Ile Thr Val Ser
1250 1255 1260
Gln Asp Ser Ile Thr Ile Gly Ser Thr Thr Val His Leu Arg Asn Gln
1265 1270 1275 1280
Asp Phe Asp Cys Leu Gln Ile Val Met Ser Arg Glu Glu Leu Leu Gln
1285 1290 1295
Val Asp Leu Ala Ser Arg Gln Ala Arg Val Leu Ser Glu Asp Ala Ser
1300 1305 1310
Lys Trp Leu Asp Thr Lys Glu Thr Ile His Glu His Leu His Lys Leu
1315 1320 1325
Ala Glu Glu His Arg Leu Leu Gly Gln Phe Val Val Val Asn Asn Tyr
1330 1335 1340
Ser Val Gln Glu Gln Ser Asp Ile Gly Thr Leu Tyr Arg Gly Val Gly
1345 1350 1355 1360
Arg Ala Tyr Tyr Asp Ala Ala His Asp Arg Met Leu Tyr Thr Asp Val
1365 1370 1375
Glu Asp Glu Thr Leu Thr Arg Asp Ala Val Leu Ala Gly Val Thr Gly
1380 1385 1390
Glu Asp Ala Tyr Phe Tyr Asn Val Lys Gln Asn Ala Leu Trp Arg Val
1395 1400 1405
Asn Ala Gly Thr His Gln Val Ile Thr Lys Tyr Tyr Pro Thr Val Leu
1410 1415 1420
Glu Asp Pro Asp Ser Lys Met Ala Arg Val Glu Gln His Gly Asn Ile
1425 1430 1435 1440
Val Val Met Ala Met Ser Arg Leu Asn Gln Pro Asp Lys Gly Val Asp
1445 1450 1455
Gly Ile Ile Tyr Leu Val His Gln Glu Ser Met Val Leu Thr Ala Val
1460 1465 1470
Ile Gly Asn Ser Glu Met Leu Lys Arg Leu Arg Gln Thr Ser Thr Ile
1475 1480 1485
Ser Leu Thr Glu Leu Arg Gly Val Asn Asp Met Ala Leu Val Ser Thr
1490 1495 1500
Met Asp Lys Leu Pro Gly Gln Lys Val Val Ala Thr Asn Ala Glu Phe
1505 1510 1515 1520
Val Thr Ile Leu Ala Lys Asp Glu Gln Gly Ser Gln Val Cys Phe Trp
1525 1530 1535
Leu Arg Thr Val Asp Gly Val Leu Ile Lys Pro Ser Ile Gln Pro Pro
1540 1545 1550
Ile Pro Thr Asp Leu Ala Leu Thr Val Ser Ser His Lys Val Ala Gly
1555 1560 1565
Gln Asp Val Phe Tyr Phe Cys Ser Ile Lys Glu Gln Thr Val Tyr Arg
1570 1575 1580
Gln Gln Gly Thr Gly Gln Thr Asn Asp Thr Ser Val Gly Ser Ile Pro
1585 1590 1595 1600
Ile Gln Gly Leu Ser Asn Leu Phe Thr Val Tyr Gly Glu Val Phe Ala
1605 1610 1615
Thr Thr Asp Asp Gly Arg Ile Leu Arg Leu Leu Gly Asp Gly Ser Thr
1620 1625 1630
Tyr Leu Glu Gly Ile Asn Glu His Trp Leu Ile Lys His Pro Lys Trp
1635 1640 1645
Trp Gln Asp Leu Ala Thr Leu Glu Ala Glu Thr Ala Thr Thr Ile Ala
1650 1655 1660
Val Phe Gly Val Arg Asp Pro Gln Asn Ala Val Val Pro Ile Trp Tyr
1665 1670 1675 1680
His Ser Gly Lys Ile Ala Val Ala Ser Ser Lys Leu Tyr Gly Lys Gln
1685 1690 1695
Leu Gln Phe Leu Gly Phe His Ser Asn Asp Gln Ala Trp Leu Phe Asp
1700 1705 1710
Leu Asp Gly Gly Ile Leu Tyr Ser Gln Pro Leu Val Asp Arg Gln Thr
1715 1720 1725
Val Asp Thr Ile Phe Ser Leu Asp Gly Lys Leu Ile Ala Pro Asp Leu
1730 1735 1740
Ile Pro Gln Val Gln Gln Met Leu Ser Gly Gln His Leu Arg Asn Pro
1745 1750 1755 1760
Val Ile Val Asp Gly Arg Leu His Val Thr Thr Ile Asp Gly Val Val
1765 1770 1775
Val Gly Phe Asp Lys Lys Ser Pro Thr Leu Val Ala Val Asn Lys Ala
1780 1785 1790
Trp Gln Ile Ser Arg Gln Asn Arg Leu Glu Glu Ala Leu Gly Asn Leu
1795 1800 1805
Leu Ser Asn Trp Ser Asp His Asn Asp Ala Ile Val Leu Gln Gly Asp
1810 1815 1820
Pro Glu Ser Ala Pro Ala Trp Tyr Tyr Val Pro Ser Arg Glu Ile Val
1825 1830 1835 1840
Thr Ala Asn Gly Leu Thr Trp Ala Asp His Pro Leu Trp Leu Gly Met
1845 1850 1855
Ala Val Gly Ala Ala Thr Pro Tyr Ile Tyr Asn Thr Ser Pro Arg Arg
1860 1865 1870
Arg Ser Ile Val Pro Glu Glu Ile Thr Ile Pro Thr Leu Ala Gly Ile
1875 1880 1885
Asp Phe Ala Leu Val Ala Ala Thr Asn Ala Thr Ile Val Ile Asp Arg
1890 1895 1900
Asp Asn Trp Lys His Tyr Lys Ala Ile Val Leu Asp Asn Lys Val Asp
1905 1910 1915 1920
Gln Asp Gly Gly Ala Lys Val Val Leu Gly Ser Glu Ile Pro Met Lys
1925 1930 1935
Val Leu Val Arg Lys Phe Gln Gly Asn Leu Glu Phe Phe Asp Pro Asn
1940 1945 1950
Asn Gly Asn Val Val Ile Leu Gln Asn Val Leu Lys Gly Asp Gly Ala
1955 1960 1965
Cys Cys Gln Ile Asp Ile Lys Val Gly Asp Ala Glu Thr Met Leu Ala
1970 1975 1980
Ser Asp Phe Ser Glu Ile Phe Ser Lys Thr His Pro Leu Val Ala Ser
1985 1990 1995 2000
Gln Val Phe Glu Leu Gly Asp Val Ile Ala Val Thr Lys His Lys Ser
2005 2010 2015
Asp Tyr Glu Asp Leu Trp Ala Ile Leu His Met Thr
2020 2025
<210> 16
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
<210> 17
<211> 93
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gggcggtacc ccgggttcga acactagtta attaagacgc gt 42
<210> 18
<211> 16379
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 60
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 120
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 180
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg gctagagcag 240
cttgccaaca tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc 300
tcagaagacc aaagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc 360
ggattccatt gcccagctat ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc 420
acctacaaat gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctgccgac 480
agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca 540
accacgtctt caaagcaagt ggattgatgt gataacatgg tggagcacga cactctcgtc 600
tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga gacttttcaa 660
caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg tcacttcatc 720
aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga taaaggaaag 780
gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg 840
agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat 900
atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga ccttcctcta 960
tataaggaag ttcatttcat ttggagagga cacgctgaaa tcaccagtct ctctctacaa 1020
atctatctct ctcgagtcta ccatggctca gatcaggtcg atggctcagg gcattcagac 1080
gctgtcgctc aactcctcta atctctccaa gacgcagaag gggccgctcg tgtcgaactc 1140
tctcttcttc ggcagcaaga agctgacaca gatctctgcc aagtcactgg gggttttcaa 1200
gaaggactcg gtgctccggg tggtccgcaa gtccagcttc cgcatctcag cttccgtcgc 1260
tacagctgag gctgaggccg tgatcgctga ggtctccact cagctcagcg aggttgtggg 1320
cgtgatcgag aggcacctgg agccaaccct cctggctgtc catctctacg ggtcagcggt 1380
tgatggtggc ctgaagcccc actccgacat cgatctcctg gttacagtga ctgtccggct 1440
ggacgagacc acgaggaggg ctctcattaa cgatctcctg gagaccagcg cttcgccagg 1500
cgagtccgag atcctcaggg cggttgaggt gacgattgtc gttcatgacg atatcattcc 1560
atggaggtac ccagctaaga gggagctcca gttcggcgag tggcagcgca atgacatcct 1620
ggccgggatt ttcgagccag cgacaatcga cattgatctg gctatcctcc tgactaaggc 1680
tagggagcac tccgtcgctc tggttggccc tgctgctgag gagctcttcg acccagtccc 1740
tgagcaggat ctcttcgagg ccctgaacga gaccctcacg ctgtggaatt ctccgcccga 1800
ctgggctggc gatgagagga atgtggtcct caccctgtcg cgcatctggt actctgctgt 1860
cacggggaag attgctccaa aggacgtggc tgctgattgg gcgatggaga ggctgccagc 1920
tcagtaccag cctgtgatcc tcgaggctag gcaggcttac ctgggccagg aggaggacag 1980
gctcgcgtcc cgggctgatc agctggagga gttcgttcat tacgtcaagg gcgagattac 2040
taaggtcgtt ggcaagtgac tcgagtttct ccataataat gtgtgagtag ttcccagata 2100
agggaattag ggttcctata gggtttcgct catgtgttga gcatataaga aacccttagt 2160
atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat 2220
ccagtactaa aatccagatc ccccgaatta attcggcgtt aattcagtac attaaaaacg 2280
tccgcaatgt gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc 2340
caccagccag ccaacagctc cccgaccggc agctcggcac aaaatcacca ctcgatacag 2400
gcagcccatc agtccgggac ggcgtcagcg ggagagccgt tgtaaggcgg cagactttgc 2460
tcatgttacc gatgctattc ggaagaacgg caactaagct gccgggtttg aaacacggat 2520
gatctcgcgg agggtagcat gttgattgta acgatgacag agcgttgctg cctgtgatca 2580
ccgcggtttc aaaatcggct ccgtcgatac tatgttatac gccaactttg aaaacaactt 2640
tgaaaaagct gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt 2700
cgtcttgtta taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa 2760
taataaatgg ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc 2820
tgcgtaaaag atacggaagg aatgtctcct gctaaggtat ataagctggt gggagaaaat 2880
gaaaacctat atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa 2940
cgggaaaagg acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtcctgcac 3000
tttgaacggc atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtcctttgc 3060
tcggaagagt atgaagatga acaaagccct gaaaagatta tcgagctgta tgcggagtgc 3120
atcaggctct ttcactccat cgacatatcg gattgtccct atacgaatag cttagacagc 3180
cgcttagccg aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac 3240
tgggaagaag acactccatt taaagatccg cgcgagctgt atgatttttt aaagacggaa 3300
aagcccgaag aggaacttgt cttttcccac ggcgacctgg gagacagcaa catctttgtg 3360
aaagatggca aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg 3420
tatgacattg ccttctgcgt ccggtcgatc agggaggata tcggggaaga acagtatgtc 3480
gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt 3540
ttactggatg aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt 3600
tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 3660
tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 3720
ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 3780
ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 3840
gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3900
aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 3960
ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4020
agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4080
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4140
aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4200
ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4260
cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4320
tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 4380
accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 4440
cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 4500
gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 4560
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 4620
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 4680
catcaccgaa acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac 4740
ggcgcggctt gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg 4800
ccagcggccg cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg 4860
gtaggcgctt tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg 4920
cgggttttaa gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc 4980
tcttttatat cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca 5040
atgtacgggt tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca 5100
ggaaagagac cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc 5160
gtacattagg aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg 5220
atcgggccag cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg 5280
atcagcttgc gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg 5340
tagatcgtct tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg 5400
tagagaaaac ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg 5460
tccgggttct tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg 5520
tactccggcc gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc 5580
tcggataccg tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc 5640
gtggtgttta accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct 5700
cgccggcaga acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct 5760
cccttccctt cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc 5820
aggtcgtaat cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg 5880
tctggaagct cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa 5940
acggccacgt ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg 6000
aaaaaatctg gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc 6060
ggcggttgcc gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg 6120
cgtgcttctg cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc 6180
aggtcatcac ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgtcacgc 6240
cgattcctcg ggcttggggg ttccagtgcc attgcagggc cggcagacaa cccagccgct 6300
tacgcctggc caaccgcccg ttcctccaca catggggcat tccacggcgt cggtgcctgg 6360
ttgttcttga ttttccatgc cgcctccttt agccgctaaa attcatctac tcatttattc 6420
atttgctcat ttactctggt agctgcgcga tgtattcaga tagcagctcg gtaatggtct 6480
tgccttggcg taccgcgtac atcttcagct tggtgtgatc ctccgccggc aactgaaagt 6540
tgacccgctt catggctggc gtgtctgcca ggctggccaa cgttgcagcc ttgctgctgc 6600
gtgcgctcgg acggccggca cttagcgtgt ttgtgctttt gctcattttc tctttacctc 6660
attaactcaa atgagttttg atttaatttc agcggccagc gcctggacct cgcgggcagc 6720
gtcgccctcg ggttctgatt caagaacggt tgtgccggcg gcggcagtgc ctgggtagct 6780
cacgcgctgc gtgatacggg actcaagaat gggcagctcg tacccggcca gcgcctcggc 6840
aacctcaccg ccgatgcgcg tgcctttgat cgcccgcgac acgacaaagg ccgcttgtag 6900
ccttccatcc gtgacctcaa tgcgctgctt aaccagctcc accaggtcgg cggtggccca 6960
tatgtcgtaa gggcttggct gcaccggaat cagcacgaag tcggctgcct tgatcgcgga 7020
cacagccaag tccgccgcct ggggcgctcc gtcgatcact acgaagtcgc gccggccgat 7080
ggccttcacg tcgcggtcaa tcgtcgggcg gtcgatgccg acaacggtta gcggttgatc 7140
ttcccgcacg gccgcccaat cgcgggcact gccctgggga tcggaatcga ctaacagaac 7200
atcggccccg gcgagttgca gggcgcgggc tagatgggtt gcgatggtcg tcttgcctga 7260
cccgcctttc tggttaagta cagcgataac cttcatgcgt tccccttgcg tatttgttta 7320
tttactcatc gcatcatata cgcagcgacc gcatgacgca agctgtttta ctcaaataca 7380
catcaccttt ttagacggcg gcgctcggtt tcttcagcgg ccaagctggc cggccaggcc 7440
gccagcttgg catcagacaa accggccagg atttcatgca gccgcacggt tgagacgtgc 7500
gcgggcggct cgaacacgta cccggccgcg atcatctccg cctcgatctc ttcggtaatg 7560
aaaaacggtt cgtcctggcc gtcctggtgc ggtttcatgc ttgttcctct tggcgttcat 7620
tctcggcggc cgccagggcg tcggcctcgg tcaatgcgtc ctcacggaag gcaccgcgcc 7680
gcctggcctc ggtgggcgtc acttcctcgc tgcgctcaag tgcgcggtac agggtcgagc 7740
gatgcacgcc aagcagtgca gccgcctctt tcacggtgcg gccttcctgg tcgatcagct 7800
cgcgggcgtg cgcgatctgt gccggggtga gggtagggcg ggggccaaac ttcacgcctc 7860
gggccttggc ggcctcgcgc ccgctccggg tgcggtcgat gattagggaa cgctcgaact 7920
cggcaatgcc ggcgaacacg gtcaacacca tgcggccggc cggcgtggtg gtgtcggccc 7980
acggctctgc caggctacgc aggcccgcgc cggcctcctg gatgcgctcg gcaatgtcca 8040
gtaggtcgcg ggtgctgcgg gccaggcggt ctagcctggt cactgtcaca acgtcgccag 8100
ggcgtaggtg gtcaagcatc ctggccagct ccgggcggtc gcgcctggtg ccggtgatct 8160
tctcggaaaa cagcttggtg cagccggccg cgtgcagttc ggcccgttgg ttggtcaagt 8220
cctggtcgtc ggtgctgacg cgggcatagc ccagcaggcc agcggcggcg ctcttgttca 8280
tggcgtaatg tctccggttc tagtcgcaag tattctactt tatgcgacta aaacacgcga 8340
caagaaaacg ccaggaaaag ggcagggcgg cagcctgtcg cgtaacttag gacttgtgcg 8400
acatgtcgtt ttcagaagac ggctgcactg aacgtcagaa gccgactgca ctatagcagc 8460
ggaggggttg gatcaaagta ctttgatccc gaggggaacc ctgtggttgg catgcacata 8520
caaatggacg aacggataaa ccttttcacg cccttttaaa tatccgttat tctaataaac 8580
gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 8640
aaggcgggaa acgacaatct gatccaagct caagctgctc tagcattcgc cattcaggct 8700
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 8760
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 8820
ttgtaaaacg acggccagtg ccaagctaag cttgcatgcc tgcaggtccc cagattagcc 8880
ttttcaattt cagaaagaat gctaacccac agatggttag agaggcttac gcagcaggtc 8940
tcatcaagac gatctacccg agcaataatc tccaggaaat caaatacctt cccaagaagg 9000
ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa gaacacagag aaagatatat 9060
ttctcaagat cagaagtact attccagtat ggacgattca aggcttgctt cacaaaccaa 9120
ggcaagtaat agagattgga gtctctaaaa aggtagttcc cactgaatca aaggccatgg 9180
agtcaaagat tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca 9240
tacagagtct cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg 9300
acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg 9360
agacttttca acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct 9420
gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg 9480
ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc 9540
cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 9600
attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 9660
acccttcctc tatataagga agttcatttc atttggagag aacacggggg actctagagg 9720
atcgcgaggc gcgccgtcga catggactac aaggacgacg atgataaggg catggactac 9780
aaggacgacg atgataaggg catggactac aaggacgacg atgataaggg cggtaccccg 9840
ggttcgaaat ggttatggta attgactatc caagtctcag agcttcgtta tatgccacta 9900
agagtgcggt cccatgtcgc ccgcttccgg gcaatccaga gcgggtgtcg cgccgggtat 9960
cacgcagagg cgcgggagag atcgggtccc atgcagattt ctggatctcc cgcgccaagg 10020
catccgccat ccggattttc ccgcgtcaag agggtccgct ccgtgaatcc cggagaagat 10080
tctatcttct ccgcgaagcg gtgttcaccg gccgcaggac cgcctgggcc gctaatccag 10140
cggcccaacg ggcagcatgg gaccgcaaga gatccccttt cttcggaagg tcttccgatg 10200
atattcattc attcagcaag aatccagaag aattcttgac tcaaaacact ctcagctgca 10260
gtttattaac tcgcaccgct cagaccgtca caaaaagccc agtattgccc gcccaaggtt 10320
gggtcaaagt gggtcgtgtg ccaggaagag gagacgctct ctttgttgag ctggttgccg 10380
agaaatcaga tgagacagtg aaggcgtact tacttggcta caatggtaag aacgactccc 10440
ccgagccggc atacgttgat attccaaaga ggggcgccga gaacggaaca tttttgttta 10500
ccgtggatct ctcgggctgc tccgttgttg tgacacagtt ggacgagcag acataccgtg 10560
ttttccacga caaacgggtg aacagctcca ttctgtacga caacgtcgta atgtatgtgg 10620
atttcaatca ctaccgctat cccagagccc acggacacgg gagagatacg ggacaagcgg 10680
tcgtctgtat gcaattcgcc gacggagtgt ggaaattgtt tctccagcgg cacgagacgg 10740
cgaattctct cccaaagata tggtcccgtg gcgatggcaa accggagatt ttcagcgacg 10800
ccgaaccccg tccacatatg gaagatggca taaaggaatt tctcaatcgg cgtcgagctt 10860
tgcaagacga attgaagcac ttcgccaaca cgcttgaagt ggacccggac atagtgtcca 10920
acgctgtcga cggcgaatac gaaggcggcc agttttccag gaacgacgat gcaattcgtg 10980
gatggaacga attacgtgca agcatgagac agaaaatggc tgaaaggaag aaactgttgc 11040
aaacaaagaa aggtatgcta gactcattca acaacccgga tcaccccctc tataaatttc 11100
gcaaacaagt gctcgacgag ctcaaggaaa tgagtgcata cgacaacacc ttggaggcat 11160
ccgagattgc ggaccgtact tggctgtggt tggagatcaa aaaagccaaa ggtacgaatg 11220
cagtggtaaa cagtggagca gccccgaagc agccgagtct taccgttggt gaacgctacg 11280
cagagatgga ggcggccctt tggatgcgta gcgacacagc ggcgtttgag caagggtatg 11340
aatcttatca aaccgttcgc gtgccgggtt acaatgaaga tatgacatcc attgcaatga 11400
agaagttgtt cgttgagagc agtctgactg acgagcagct gggcgccttg tgcaagcgta 11460
tatccattgc tagcgatcaa gagaaccatc ttctgacaca gaagacgcag gagattgaga 11520
gcactttcaa acaattttca gatggcagct ttttggagtc ggcgcctcag gatttcttcc 11580
tgagacgagt cggtgataaa tctggaggac gctgctatcc tcttgtaagg gtaatggccg 11640
tcgcactgtc tgaaggagaa gacgttgccc agaattttat ggagaacctg aggacattgt 11700
ctaggaatcc agagtattca ggcgaatcgg caagttcaca agccacaaat gcagggttgt 11760
tcagagatgc cctggccggc ctgcacgcga atgtggaagc cattgccgca tctgagaaac 11820
tacctgctgt gtttggactg cccgatgttg ctacaatgtt gagccaagcg agcgaaagca 11880
aggtttacgc tttgaatacc gaaacacaca gcatgctggt tgcggtgatt gtagacaaac 11940
agagtctggc gcgcaagtat tacttctatg atcccaactt tctcatggcc ggtttctccg 12000
acataactcg tttctcggaa gccctcaagt atcactttgt gaatctccag ttcgccggaa 12060
actacaaagc gtttggcaca gcggaggctc cccagtttaa cctggtcctt attgatacga 12120
ccgccatgga ggatgcgcat gttgacggag ggatcaccgt gaaacaattg agcgagggcg 12180
ttgatcttac taatgtcttt gacagcacca aaaaggctcg tgtggtaact gaggcagcag 12240
ctcgcttggc tgaagaccaa caactgcgca ctggcctcac cataatagag gctgaccagt 12300
gggcaaacag gtggcaagat gccgtcatac gattgaaact caaggaagga ttaagtggag 12360
agtggatgcc attgctggag aaaatgcaag aaaacgcaga tggttcttat cagattgatt 12420
tcatcaacac caagaatact ggggaaatcc gtggggtttc cactagagat gcgacgttca 12480
aagaattcaa atcctacctg gataaaaatt tagacgtatt gcataaggat ttcatctttc 12540
aggatggagt tttcaaacca ctggagggag agcccattga cgggttgaat gcaatgttcg 12600
cggtccaaat gattatagat tggttcaacc gggggactaa tgctgaggaa gatgcggcct 12660
caggatccac caatgcccaa ctttccctag cgattaaagt gcacagctac attaacatgg 12720
cccaagtagc gcatactgca gttacagatg ttgccaaggt atacaacctt gtaaagtcag 12780
cactgtatgg cgagcaggta gctggggaga catcgctgtt cacatttggg aaggcgttga 12840
aatttgcagc aacagaaggg ttgggagtct tactaggggc gtcctttgtg atcttggatg 12900
cctaccagct gtccctcgtg gaaaacgatg tgcagaaggc cattatcggc acacaactag 12960
cctttgattc cgcaagcttt ctagcgggta ttggtgcaat tgttgctgga gcatcgggcg 13020
tgggcatgat tttgggcgga gctggcgtgg tattgggagg cctgggagtt ggctttggag 13080
ccttagctca ggcattcggg gaaatcacag cagacgccca actggtgggc aagtacttcg 13140
cactcttgga cgaggcatac cgcggtggcg gatacagata cgaatctaaa gacaaggcat 13200
tgatgcctct tgcaggagca gtcatcgaca aaatacgact ggccgacaaa catctccaag 13260
ttcatttcga cagccaattc atatatcgaa cccatcatgg cagcacagga tccggcaaaa 13320
ttaattactt cttctgggct ggcgacatgc cgcagatggt ccacgatcgc tcccaagcta 13380
tcaatgttcg agagcaaatt gggtataaga acgataatgc tactttggag ctaccggagg 13440
atgcctctga tctgatcctg cctgccacgc ccaagtcatt tatcacttac cgatacgaga 13500
ttctaccatt cgctacttcc cgccacgata cgggtttcga tgtcattaga cgcctggaag 13560
aagactacag attcgattac gatttctaca tcttcccatc tgaatacaca atcagaacca 13620
tcaaacacga gtacgtagaa acaaaggtgg cagtagtcct ggataggcgc tctttgcgac 13680
tgggagcacc tgagcttctc aaggagcaac atggctattt gatctatgag atcgagggga 13740
ccggaggaca atacgttata tgtcttaacc agggatccac tgtcacactg aagaccaccg 13800
gatcagaggc aaataaaaca ttatggatac ttgatgcccg acaactcatt gcaggcaacg 13860
attcaatcac ggtctctcaa gatagcatca ccatcggcag cacaacagtc catctccgta 13920
accaagattt cgattgccta cagatcgtca tgagtcggga ggagcttctc caagtggact 13980
tggcgtctgg agaagcccgc gtgttttcag aagatgccag taaatggctc gacaccaaag 14040
aaacaatcca tgaacatctc cataagttgg ctgaagaaca caggttgcat ggccagtttg 14100
tagtggtgaa caattattat cgtggagtgg gccgtgccta ctatgatgct gctcacgatc 14160
ggatgttgta tacagatgtg gaagatgaaa ctctgactcg ggatgctgtt cttgccggcg 14220
ttacaggcga agatgcctac ttctacaatg taaagcagaa tgccctgtgg cgagtgaatg 14280
caggcacaca ccaagtaata accaaatact atccgacgtt actggaggac cctgacagca 14340
acatggcaag agttgagcag catggcaaca tcgtcgtgat ggccatgagc aggctcaacc 14400
acccggacaa gggagtaaat ggaatcatct atctcgttca tcaagaaagc atcgtgctta 14460
ctgctgtgat cggcaattca gagatgctca aacgtcttcg ccaaacgagt acaattagct 14520
taacagaatt gcgaggtgtc aatgacatgg ctctggtttc cacgatggac agattacccg 14580
gccaaaaggt tgtcgctacc aacgctgagt ttgtgacact tctggctaag gatgagcaag 14640
gagccaatat caaggagcag acggtctatt gccaacaagg tacgggccag accaacgata 14700
catctattga aagcatcccg attccagggc tctctaatct tttaaccgtg tacggtgaag 14760
tattcgctac aactaatgat ggtcgcatcc ttcgcctgct tggggacggc tcaacatgtc 14820
tcgagggcat caatgagcat tggctcatca agcatccaaa atggtggcag gacttggcta 14880
ccttggaggc agagaaagcc acgacagttg cagtattcgg tgttagagac ctacaaaatg 14940
cagtggtgcc aatttggtac cacaacggaa agatcgcggt ggcctcatca aaactgtatg 15000
gcaaacaact gcaatttttg gggtttaaca gcaacggtga agcttggttg ttcgacttgg 15060
acgacggcat tctgtatagc cagccgcttg tggatagctt tctgtatgca atctttagtt 15120
tggacgggaa gcttattgca ccaatcttta ttccacaagt ggagcaaatg ctcagcggcc 15180
agcatctcag aaaccctgtc atcgtggacg gccgtttgca tgtgactacc atagacggtg 15240
ttgttgttgg atttgacaag aagtcgccaa ctctggttgc agtgaacaag gcctggcaga 15300
tatctcgtca gaaccgatta gaagaggccc tcggaaacct gctgtccaat tggtctgatc 15360
acaatgatgc gattgtgttg cagggtgatc cagaatcagc gccggcatgg tattatgttc 15420
catcaagaga aatagtgacc gccaatggtc taacatgggc ggatcatcct ctatggctag 15480
gaatggctgt cggtgctgca acgccataca tctacagcac atcccgacgc cgtgtgtatg 15540
ttgtggagag caaaaacatg gccaaagaca aaggagaatt cgcagcagca cattgctttg 15600
caaaatcact tgtgctacag agcatcgttc ccgaggaaat aacgattccg acgctggctg 15660
gcatcgattt cgcactcgtt gcagcgacaa atgcaaccat tgtaatagac cgagacaatt 15720
ggaagcatta caaggccatt gtgctcgaca acaaggtgga tcaggacggt ggcgctaagg 15780
tggtagtcgg cagtgagatt cccatgaaag ttctggtgcg caaatttcag ggaaatctgg 15840
agttcttcga tccaaacaat ggtaatgttg ttatactcca gaatgtgttg aagggagacg 15900
gagcttgttg ccagatagag atcaaagtag gcgatgctga gactatgctt gtatccgatt 15960
tctcggagat attctcaaag acgcatcctc tggtggcaag ccaagtattt gagcttgggg 16020
atgtgattgc agtcacaaaa cacaaatcgg actatgcgga tctgtgggcc attctacgca 16080
tgacatagac gcgtcttaat taactagtga gctcgaattt ccccgatcgt tcaaacattt 16140
ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat 16200
ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga 16260
gatgggtttt tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa 16320
tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcgg 16379
<210> 19
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19

Claims (11)

1. The insect-resistant cycadoxin gene is characterized in that the nucleotide sequence of the insect-resistant cycadoxin gene is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
2. A codon optimization by the pest-resistant thifenuron gene of claim 1 using synonymous codons instead of rare codons depending on codon preferences of different plants or crops; the invention uses synonymous codons to replace rare codons to obtain the cycadoxin optimized gene according to the preference of cotton codons, and is characterized in that the nucleotide sequence of the cycadoxin optimized gene is SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.
3. A protein encoding the insect-resistant thifenuron protein gene as claimed in claim 1, wherein the amino acid sequence of the protein is SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No. 15.
4. A method for obtaining related toxic genes by using software or websites to perform homology comparison in different species by taking the nucleotide sequence of the cycotoxin protein gene as a target, wherein the obtained nucleotide sequence has the sequence similarity of more than 70 percent; a nucleotide sequence or a functionally similar subfragment that has an amino acid sequence similarity of greater than 50% to the thaumatotoxin protein gene of any one of claims 1 and 2.
5. A pesticidal use or manufacture of a pesticide encoding the pest-resistant cyhalothrin gene of any one of claims 1, 2, including but not limited to plant or crop pests such as cotton bollworm, plutella xylostella, spodoptera frugiperda, and the like.
6. A plant or crop expression vector constructed by using the insect-resistant cyhalothrin gene according to any one of claims 1 and 2, wherein the plant expression vector is constructed by a method comprising:
preparation of a linearized vector: using restriction enzymes BstBI and MluI to double-enzyme-cut VM062-3 FLAG vector to linearize the annular vector;
amplification of target sequences with homology arms:
with the forward primer:
5'-gggcggtaccccgggttcgaaATGGTTATGGTAATTGACTATCCAAGTC-3', respectively; reverse primer:
5'-cactagttaattaagacgcgtCTATGTCATGCGTAGAATGGCCC-3' is a primer, and PCR amplification is carried out on the cyathrin gene;
and (3) detecting and recovering a vector enzyme digestion product and a PCR product: agarose gel electrophoresis is used for detecting the target gene fragment and the carrier enzyme digestion product
Figure FDA0003571196020000021
Recovering a target fragment from the Quick Gel Extraction Kit;
glue recovery product recombination: use of
Figure FDA0003571196020000022
-Basic Seamless Cloning and Assembly Kit for homologous recombination in the following reaction system: 2 × Basic Assembly Mix 5 μ L; carrying out enzyme digestion on the vector X mu L; mu.L of PCR product; sterile water was added to 10 μ L. In a 10 μ L reaction system, the addition amount of the vector and each insert is 0.01-0.25 pmols, and the optimal molar ratio of the vector to each insert is 1: 2; lightly mixing, reacting at 50 deg.C for 15min, and converting;
sequencing and identifying: positive clones were selected by colony PCR and plasmid PCR for sequencing analysis.
7. The method of claim 6, wherein the amplification system is: KOD One TM PCR Master Mix 25. mu.L; upstream primer 10. mu.M each 1.5. mu.L; downstream primer 10. mu.M each 1.5. mu.L; 2 mu L of template; 20 mu L of sterilized water; the total volume is 50 mu L;
the PCR program is 98 ℃ for 5 min; 30 cycles of 98 ℃ for 30s, 50 ℃ for 30s and 68 ℃ for 3 min; storing at 68 deg.C for 10min and 4 deg.C.
8. The method of claim 6, wherein the transformation comprises:
(1) thawing the Trans1-T1 competent cells on ice, standing for 2-5 minutes, observing whether the cells are thawed, and flicking the bottom of the tube 1-2 times by using fingers; adding 2 μ L of recombinant product into 50 μ L of cells, bouncing the tube wall of the centrifuge, mixing well, and standing on ice for 30 min;
(2) immediately transferring to ice to cool for 2min after heat shock is carried out for 30s in 42 ℃ water bath; adding 450 μ L of normal temperature non-resistant LB liquid culture medium, and culturing in 37 deg.C shaking table at 250rpm for 1 h;
(3) the cells were centrifuged at 5000rpm for 2min, collected, 100. mu.L of the liquid was resuspended, spread evenly on LB solid plates containing kanamycin resistance, and cultured overnight in a 37 ℃ incubator.
9. A method of combating a pest in a plant or crop using a pest-resistant threonin gene according to any one of claims 1 and 2, wherein said method of combating a pest in a plant or crop comprises:
introducing a plant expression vector containing a target sequence into a plant cell, and thereby obtaining a transgenic plant resistant or capable of killing pests and progeny thereof, including seeds and plant tissue of any part; or the protein is obtained by homologous or heterologous expression and applied to plants or crops;
wherein the plants or crops include, but are not limited to, cotton, corn, wheat, and rice.
10. A method for detecting the expression of a periferritin gene of a transgenic cotton plant according to claim 9, wherein the method for detecting the expression of the periferritin gene of the transgenic cotton plant comprises the steps of taking leaves of a homozygous transgenic cotton strain, extracting total protein of the leaves, and using the total protein for Western Blot detection; the method specifically comprises the following steps:
(1) taking 1-2 g of a fresh sample, and grinding the sample in a mortar by using liquid nitrogen; grinding, placing in a 10mL test tube, adding protein extract, and shaking at 4 deg.C for 30 min;
(2) centrifuging at 12000rpm at 4 deg.C for 20 min; extracting the supernatant, and completing sample preparation;
(3) and taking the supernatant, adding a Loading Buffer, boiling for 5-10 min at 98 ℃, and determining the protein expression condition by using an SDS-polyacrylamide gel electrophoresis method.
11. A method of detecting ferritin gene expression from a transgenic cotton plant according to claim 10 wherein the protein extract comprises 0.01M PBS pH 8, 1mM PMSF, 2mM DTT.
CN202210324163.XA 2022-03-29 2022-03-29 Insect-resistant threo-ferritin gene, expression vector and application Active CN114853858B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210324163.XA CN114853858B (en) 2022-03-29 2022-03-29 Insect-resistant threo-ferritin gene, expression vector and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210324163.XA CN114853858B (en) 2022-03-29 2022-03-29 Insect-resistant threo-ferritin gene, expression vector and application

Publications (2)

Publication Number Publication Date
CN114853858A true CN114853858A (en) 2022-08-05
CN114853858B CN114853858B (en) 2023-09-15

Family

ID=82628976

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210324163.XA Active CN114853858B (en) 2022-03-29 2022-03-29 Insect-resistant threo-ferritin gene, expression vector and application

Country Status (1)

Country Link
CN (1) CN114853858B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1818067A (en) * 2006-02-27 2006-08-16 浙江大学 Zoophobous fusion protein and use thereof
CN102031266A (en) * 2010-03-25 2011-04-27 浙江大学 Insect-resistant fusion gene, fused protein and application of fused protein
CN103501614A (en) * 2011-05-31 2014-01-08 先正达参股股份有限公司 Pesticidal mixtures including isoxazoline derivatives
US20170039316A1 (en) * 2003-11-12 2017-02-09 Yuriy Fofanov Compositions, processes and algorithms for microbial detection
CN107205400A (en) * 2015-04-16 2017-09-26 龙灯农业化工国际有限公司 Synergistic insecticidal compositions containing anabasine insecticide, pyrethroid insectide and abamectin insecticide
CN107920530A (en) * 2015-08-25 2018-04-17 巴斯夫欧洲公司 The pest in cotton is prevented by ginkgolides and Bilobalide
CN109924210A (en) * 2017-12-15 2019-06-25 江苏龙灯化学有限公司 The composition for killing harmful organism comprising thiodicarb and imidacloprid

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170039316A1 (en) * 2003-11-12 2017-02-09 Yuriy Fofanov Compositions, processes and algorithms for microbial detection
CN1818067A (en) * 2006-02-27 2006-08-16 浙江大学 Zoophobous fusion protein and use thereof
CN102031266A (en) * 2010-03-25 2011-04-27 浙江大学 Insect-resistant fusion gene, fused protein and application of fused protein
CN103501614A (en) * 2011-05-31 2014-01-08 先正达参股股份有限公司 Pesticidal mixtures including isoxazoline derivatives
CN107205400A (en) * 2015-04-16 2017-09-26 龙灯农业化工国际有限公司 Synergistic insecticidal compositions containing anabasine insecticide, pyrethroid insectide and abamectin insecticide
CN107920530A (en) * 2015-08-25 2018-04-17 巴斯夫欧洲公司 The pest in cotton is prevented by ginkgolides and Bilobalide
CN109924210A (en) * 2017-12-15 2019-06-25 江苏龙灯化学有限公司 The composition for killing harmful organism comprising thiodicarb and imidacloprid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THOMAS E. MARLER: "Boomeranging in structural defense Phytophagous insect uses cycad trichomes to defend against entomophagy", 《PLANT SIGNALING & BEHAVIOR》, vol. 7, pages 1 - 4 *

Also Published As

Publication number Publication date
CN114853858B (en) 2023-09-15

Similar Documents

Publication Publication Date Title
CN111171118B (en) Plant insect-resistant gene mCry2Ab, and vector and application thereof
TW202134424A (en) Methods and compositions for culturing hemoglobin-dependent bacteria
CN104313036A (en) Insect resistant gene mCry2Ab and application thereof
CN103184230B (en) Fusion protein gene TAT-sVP7 and application thereof
WO2010121533A1 (en) Rape heat shock protein gene hsp17.8 and uses thereof
CN1037913C (en) Expressive carrier with coded insect-killing protein fusion gene, and transfer gene plant
CN111041036B (en) Coding insecticidal protein insect-resistant fusion gene mCryAb-VIP3A, expression vector and application thereof
CN102161996B (en) Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants
CN102690341A (en) Plant tillering related protein and coding gene thereof
CN113493794B (en) Gene GmGRX4 with resistance to soybean mosaic virus and application thereof
CN114853858B (en) Insect-resistant threo-ferritin gene, expression vector and application
CN105111295A (en) Cultivation method of genetically modified WMYB-R wheat resistant to root rot and sheath blight and related biological material thereof
CN103275202B (en) Disease resistance-related protein RCR1 derived from wheat, related biomaterials thereof, and application for same
CN103725692A (en) Zinc finger protein gene used for controlling rice fertility
CN101845445A (en) Cre recombinase recombination gene and Cre/loxP-mediated transgenosis safe plant expression vector
CN107365372B (en) L-type agglutinin of Litopenaeus vannamei, and coding gene and application thereof
CN102776202A (en) Cultivation method of male sterile plant
CN114853860A (en) Protein related to shortening larch breeding cycle and application thereof
CN105505984B (en) Carrier of rice respiratory burst oxidase gene OsRboh (LOC_Os01g25820) and application thereof
CN110564702B (en) Chilo suppressalis growth and development related protein ND, coding gene, dsRNA interference sequence and application thereof
CN114940996A (en) Application of tomato SlULT1 gene in improving drought resistance of tomato
MXPA04010704A (en) Wound-inducible expression in plants.
CN101704884A (en) Plant drought resistance and salt tolerance associated protein EeABF6, coding gene and application thereof
CN108276481A (en) Upland cotton GhLEA3 genes and its application in terms of low-temperature resistance stress
CN104946666B (en) Rice Os ACBP5 genes and its application in Rice Resistance characteristic of disease is improved

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant