KR101613523B1 - Novel Cnidium mottle virus gene from cnidium plant - Google Patents

Abstract

More particularly, the present invention relates to a gene encoding a Cnidium mottle virus comprising the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2, A recombinant vector containing a mottle virus gene and a host cell transformed with the recombinant vector.

Description

(Novel Cnidium mottle virus gene from cnidium plant)

More particularly, the present invention relates to a gene encoding a cnidium mottle virus comprising the nucleotide sequence of SEQ ID NO: 1 derived from the genus Acanthopanax senticosus and the nucleotide sequence of SEQ ID NO: 2, A recombinant vector comprising the genus, the genomic DNA virus gene, and a host cell transformed with the recombinant vector.

The emergence and risk of emergent viruses and new viral diseases are increasing gradually due to ecosystem changes due to climate warming, evolution and variation of plants and viruses, and increased trade in agricultural products. About 1,000 plant viruses have been reported so far worldwide, and new viruses are still reported in various plants. Currently, about 100 viruses have been reported in various plants in Korea. However, detailed investigations of the viruses have been carried out only in some crops, and most crops are only fragmented. Thus, it is not yet clear which viral diseases are occurring in many major crops.

Plant diseases are abnormalities of the host cells and tissues that result from the continued influences of pathogens or environmental factors on the plant host, including abnormalities in plant morphology, physiology, etc., resulting in a reduction in the resulting economic value It is also said. Causes of these plant diseases include fungi, bacteria, Nematode, Mycoplasma, protozoa and viruses. However, the virus is a pathogen having a size of less than 1 占 퐉 and its substance can not be observed even with an optical microscope, and since it requires an electron microscope, it is difficult to diagnose a virus that has developed in a plant with the naked eye. Accordingly, various methods for diagnosing viruses have been developed and used. In particular, it is necessary to study viruses that cause disease in astragalus plants.

Korean Patent No. 10-0961156 discloses a probe set for detecting plant virus, Korean Patent No. 10-1153458 discloses a duplex primer for simultaneous diagnosis of INSV and TSWV and its use, As in the case of the invention, a new gene for the celestial mottle virus originating from the ciliate plant has not been disclosed.

The present invention has been made in view of the above-mentioned needs. The present inventors prepared a cDNA library of 138 plant samples suspected of being infected with a plant virus, and obtained PCR results of the primer set based on the contaminants thereof , Confirming that the gene is a novel Cnidium mottle virus gene having a size of about 7.3 kb and about 3.1 kb which eventually causes disease in the cinnabar line, thereby completing the present invention.

In order to accomplish the object of the present invention, the present invention provides a Cnidium mottle virus gene comprising the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ.

The present invention also provides a recombinant vector comprising the Cnidium mottle virus gene.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention relates to the isolation of a novel ciliaris mottle virus gene from a ciliotus plant whose pathology is due to a virus but whose causative virus has not yet been identified. Since the virus can be diagnosed by using the gene of the present invention, the virus can be used for the monitoring of diseases or diseases, and it can be used for disease development. Especially, And contribute to the improvement of the quality and quality of the host plants containing the ciliates.

In order to accomplish the object of the present invention, the present invention provides a Cnidium mottle virus gene derived from T. gentry plants.

The gene encoding the T. gondii virus may be a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a sequence homology of at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively May contain a base sequence. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In addition, the present invention provides a recombinant vector comprising the genus Kanttle mottle virus gene.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, the mantle virus gene sequence can be inserted into a recombinant vector. The term "recombinant vector" means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. The recombinant vector is preferably a plant expression vector.

In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

Expression vectors containing the celestial mottle virus gene sequences and proper transcription / translation control signals from the celestial plant may be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant gene, aadA gene, and the like, but are not limited thereto.

In the plant expression vector according to the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter " refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the plant expression vector according to the present invention, the terminator can be a conventional terminator such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaci And the terminator of the Octopine gene of Agrobacterium tumefaciens , but is not limited thereto.

The present invention also provides a host cell transformed with the recombinant vector.

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells. The host cell is preferably a plant cell.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl ₂ method, one method (Hanahan, D., J. MoI. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell, And an electric drilling method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

One. Sample collection and total RNA  extraction

138 plant samples suspected of being infected with plant viruses were collected and all samples were each ground using liquid nitrogen. All samples were stored at cryogenic temperatures. Total RNA was extracted after mixing the same amount of each sample in the same amount. Total RNA extraction was performed with Tri Reagent (MRC).

2. Ribosome RNA  Removal, double strand cDNA  Synthesis, library creation and sequencing

Ribosomal RNA was removed from the extracted total RNA using Ribo-Zero ™ Magnetic Kit (plant leaf) (Epicenter). The prepared RNA was disrupted with a short fragment and then synthesized with double-stranded cDNA. The synthesized double-stranded cDNA was end-repaired and an adapter was attached for the cloning required for library production. This series of procedures was performed using the TrueqRNA sample prep kit (Illumina). Inserted DNA (insert DNA) of appropriate size purified using BluePippin ™ 2% agarose gel cassette (Sage Science) was selected for PCR amplification. Finally, the size and quantity / quality of the inserted DNA suitable for library production were measured using an Agilent 2100 BioAnalyzer 2100 (Agilent Technologies). The library production was completed, and the size of the library segment was confirmed to be approximately 350-450bp. The library was sequenced using Illumina HiSeq2500 and raw data was generated at about 40 Gbp using a paired-end sequencing method.

3. Sequenced low  data( raw data ) And plant virus-related contig ( contig ) Production

After selecting high-quality data from low-level data, we obtained plant virus-related readings and created contig. This process was carried out using de novo transcriptome analysis (SG-de novo assembler). Approximately 7,000 contigs with plant virus-related sequences were obtained. When the length of the conti was too short (<500 bp), data with low coverage was removed when compared with the virus database (DataBase), and about 700 contigs were finally obtained.

4. primer  Fabrication and identification of whole genome

When compared with the virus-related database (DataBase), about 150 to 90 contigues were obtained by removing the contigus that showed about 85-90% or more homology. We confirmed the most homologous virus through each database, They were grouped into two groups, each of which had high homology to the same virus. The contigs belonging to each group aligned their plant viral genome as a reference sequence and confirmed their position.

Based on the nucleotide sequences of these contigs, primers were prepared and PCR was performed on 138 plant samples using the primers. The PCR results were confirmed by agarose gel electrophoresis or by sequencing of the banded PCR products. Based on the nucleotide sequence obtained from the PCR result of the primer set based on the contig, a primer was further prepared to obtain an additional base sequence.

Example  1. Identification of the nucleotide sequence of a new plant virus

(SEQ ID NO: 1 and SEQ ID NO: 2), the genome information was confirmed to be about the nucleotide sequence of the plant virus (+) Single-stranded RNA genome) of about 7.3 kb and about 3.1 kb, and each gene has a maximum nucleotide sequence identity of 76% and 67% Respectively. In the present invention, the name of the virus was named Cnidium mottle virus.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel Cnidium mottle virus gene from cnidium plant <130> PN14228 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 7281 <212> DNA <213> Cnidium mottle virus <400> 1 ttgaaaagca atctgcgaac tttgtcacta tttgtttcct caactagtta ttggacttat 60 ccaattccct tcgttttcgc acttactagc accgggctta tccggattct ctctttgttt 120 tcttttgttg tactgtggtc atacagtttt ccttttggaa ttcaagtctt aacttgaaca 180 acaacttttc tctttgttat ttctcttctc tagtaaccac tttaattttc tatgggttac 240 tcaagaggtt ctcgcggcaa ggctgacaaa gccaagctgt gcggcatctg ctttgcccct 300 tttcctgatg ctcaggaaag aaaggtccac tatcgaaaat ataactgccg tgactcgtcc 360 ggtcagttgg gtgggctggt tagcacaggg cgctgtgctg ggatgaagga gaagaatctg 420 caagttgaac gtgatcgatg tgccatgagg gagtttgagg aggaggagct gcttgaagct 480 gatgtggaaa tggagttcat gtcagagttg atggacttca tagccccttt tccttttcca 540 ggtatctcct ttggtactgt ttcaatgact gttcccaaaa ttgggagctc ttatgctgga 600 aggaaaattc ttaaggctaa ggggcttgat gttgaggagt acgctaagtt ccaggcaccc 660 aaggctggtg tacggaactt tcttcagccc aattgtagga ctgatgctga agttcttacc 720 acgattgtcc gcactgcaat agagaacccg ggctttggta atccttcttt tgattacaca 780 ggccttggag gggatgagtt taagtaccgt tttgataaag acggtttgtt tgcggatgat 840 gatctgggtt tggctcgttt tgtttccgct ggtttcactg agggttctag acccaacgtc 900 tttgacgtag ctcctttaac ctctggtgac tttaaggtta tgatacaagc tctttctgat 960 gaatggtccg aatggattta tttcgaggaa gattatgagg aggctctctc tgaggaattt 1020 gagatcttct ttccttttct tgagagtgct aaggtgtgtg aacccgcacc cctgagtcca 1080 acctcagtgt tggattctgc agactcctac ctgccaggca ctaagtgggt atacgattca 1140 gatgaggagg accacattcc tgttgctcca attgatcggt ttcataataa aaagtttgat 1200 ctcgattttg aagactattg cgataccgag tatctagagc agcctgctga aactgtctat 1260 acagaaacgt ggtttattga tggttgtctt ccggggccct ggtttaagag aggagtttca 1320 ttggatgaag caattatttg cgagaaatta cccttgattg gtaagagtcc actggagctc 1380 ataatacagt accaggtcaa accaactgga gacctcctcc ttgacgttca gcgagcgctc 1440 tttgcttatt atgagaagga agcctggccg aacaaaaagc actttgatct taatggggtg 1500 ccaacaatgc ttgtttttga ggattggagg tgtgatttga gatattgtct ctatgcaccc 1560 tttgttcagg gtaaccaatt gaagcatctc atggctattg ttgatgcccc tttacctggc 1620 tggcttttgg acttcccaaa gcggcagaag agtggtacac tacaagctag tggctggaat 1680 cctctccgtt acttctcaaa gcaggccaca ttgggcttcc ttgatggagt tattgccaag 1740 cttcgagagc ttttaggtcc tgtgatcaac gctgctggtt atgtctggga ccttattatc 1800 aaggccaagg attatgcctt tgctatgctg gatgaaatga tctctaaacg cggtgaaatt 1860 ctcaaggctc ttttacaacc actcttgtat gttggtggct tcttgatttt tcttgggtct 1920 cttaagggat tgcaaacaat tgttgagaag ctcggtgttc ccttagctgt tcttacggct 1980 gctgctattg gcattggtgt ttacatttta gttcagttcc ttggtcaagc acatatggga 2040 gctcttaaga gatcaaccaa aatttgggaa ctggtacaga aatgggatca accttcagaa 2100 gtcaggcgta ttgatgaaga gattgttgaa atcttggtgg aagaaagtcc agaaaaggaa 2160 gagtggtttc gcgagttaat ggcatctccc catagcatac cagaatgtgt tattgattta 2220 acacacatga aggatttact tggagggaag tgctcttcct tcaaagaagc ctttgaggtg 2280 ggcacttctg tggcatcatc tcctgggatg ctttttggtt ccggatttat ctggaaaatt 2340 ctactgttgt tgtgtcctct ttctatgttt ggtgtttcca agactctctc atgtgctaag 2400 gacctgatta ctatacaagg gggtcaagat gcagctggaa gattcttcca agatattgtt 2460 ggtggtactc aggaggtctt ttacacacta acaggtagca agagtgagtt tttggattat 2520 atctatgcca ctattggggt tgatttccaa gcttggcgct cagaggtcat ggagcttaca 2580 acagcaacac ccacttctat ttttttggga cctcaagaga gactgaaaag attgagatca 2640 tgcaaggata aggcagatag gttgatactt cagatggatt cacgcaaggt accgggagct 2700 tatatcacac attttaataa tttgctccag tctttggatc gtgccttggt cgaatgtcag 2760 caagctcttt ctgttggcaa atggcgtaag accccagctt gtatctggct ctatggggac 2820 tcccatgttg ggaaatcggt atgttcccag taccttattg atgatgtctt ggactcgctg 2880 gattatgccc agacgggcag agtgttttct cggaatggat ctgactcttt ctggtcatgt 2940 tataagaatc agagtgccgt gctgtacgac gactttggcg ctgtttcaga aggtggtcat 3000 tttgatgaag ctgaaattat aaggctcatt gcacccgctc ccttaccctt gaacatgcct 3060 aatctggagg caaaagggaa tacgtgttgc acatctgatt ttgtatttat aacagctaat 3120 caagctggtt tgacaccagg tgctgtagtt cactgcagga aggcatttga gaaccggaga 3180 ttgattttgg cggaagttac agcagttgaa ggaggtaggt atcgtgatag gtatcgtttc 3240 actttacatc aaaagaatga gccttattca cgagatgatc gatttcaagt tatgaactat 3300 gaacaattcc tccagtacac cgttaatcaa tcgaggcaac acttctcaga gcaggttgat 3360 ctccgggacg cgccacacac acagatcttt gctgcagcag atcagatagc agcagaagct 3420 gaagagggtt tccagactgc aggcataaag gtaccattca ccatgctgaa gagcttatct 3480 ggggaccagc tcacttacta ctctgaggag aagatgatgg agatcacgga gacgttgaca 3540 cctgaggatg cagatataat tcgttcacgc cttgatccag gagtcataac tcagcagttg 3600 cagatgctac ttccaaaagc tgcctatgtg aacctcaaaa atcactttgg cttaggtact 3660 gatcctttcg agcaaccaaa tccttatttt aattgttcgc agagagccaa ggtcattggt 3720 cgatttatga agccaaaagt tacagaagaa cagttagagc agcacaagcg tgggttcgta 3780 gtcatagcaa aggaactagc ccaaggagct tgcaaggcaa ttaatgatgc tccttttctg 3840 gtcaagctct tactaggttt tggagcatta tactttgttg gtctcccctt gttgagttgg 3900 ttgaaaagtc tttatactac accttgtttg ttgacgtttg ccacccttgg aactatgaaa 3960 gcctctggtt cgttgtccag ttcacaagac caggaaacac gacgtacggc atctggtaga 4020 gaaagaagac gttatttgtt ggaggcttct ggacctggtg ctgtggaagc aaaggctcaa 4080 gatgtggaat ctgaacttgc atctctatcc aaacatctca ttggtttcac gagtatagat 4140 tatcctgatc atcattatcg gggtattgct ctcggaggaa ctcgggtttt gatggtttat 4200 catgtttggt tggagttgca aagtggttgt tataaagttg gttctctgac caaaactttt 4260 cctttcaccg tcaatcggaa aaattgtagg ttccagaggc taggacttaa ggacttggtt 4320 atcgtggact ttccaccaac ctttgtttct tttcctgtgc tgaaaattga gaaatggttg 4380 ctctcttcac atgacccttt catggctggc tctggttggt ttatggaaat gttatttcga 4440 gatcatggtg ttctggaagt tgctagagag gaggctgatt acaccctgtt ggacacaaat 4500 gatgtgtacg acgcagcttt tctcaagggt gttgggttga acaagtgtgt gaggtacact 4560 atttgtgatg atactgggac aggttatcgt aatgacttct tttatgtttc ccagtgtggc 4620 actcctcttg ttgcaaatta tggcagagga aaaggcctga aaatagcttc catccacgtt 4680 gtgcaccatt tctctgcaac gcaaaaagat gccattatag ccggctctgg aagtctcatc 4740 actaaggagg aatacctgga ggcaagtctt cttttggggg aaataaaaca ccctcttgag 4800 acggatcgaa ttcaggcttc cggttgccta agtggtgagg agttttttga cgcagaaact 4860 gttttcccag aaggccttct tgaaccatcg gaagccccga ggcaagcaac cactagtgag 4920 attaggaaga gctccatatc agctgacttg gaagctctta ctggggagaa aaagaaaacg 4980 gaaccagcta ttatacataa tagggatacc cgacttcatg atcggcacct ggatattttt 5040 aaaaagggta tgctgaagta caaagctgtt gctgcggaca tgagcccagt tactgaagaa 5100 gaacaaaaga tttgggatct aacttgggat agtatctttg atcttccagg aggtattgag 5160 aagaagtgcc atctattgtc tgaggatgaa aatttaaatg gtgtcaatgg ggacaatgag 5220 taccgaggaa tggttgtctc cacaagtgag ggttggccag aggtcttgaa tcgcaagaat 5280 ggtgaagctg gcaaggagcg atttctgcta ggtttgccag ggtgctacac attgaatcga 5340 gacctaccca tgtaccagag aatactggat atggatgagt tgtgtgccac cacgataccc 5400 tgtattgttg gtttggatac tgcaaaggat gaacgtcttc ccctctccaa gatctatcaa 5460 gatgtcaaga cgaggttatt cacaatactc ccaatggagt ataattacct tgtcagaaag 5520 tattttgggt cttttgttgc tgagttgatg aagttgcata atgtcattcc cacaaaagtt 5580 ggcattaacc cacttggtta cgattggaca atactgggga agaggatgca tgccaaaggc 5640 accaattggt ttaatggaga ctattcccgc tttgatggtg ttacaccacg ttgcttactg 5700 attgagatag cacggcgtat aacgctactt tacaacgatg atcaaggaaa taggcgcctg 5760 cacctaatgt tggctgctac aactcgcctg ggtgtagcag gcataggctt atatagggta 5820 tctggtggta tcccttcagg ctttgccctt actgtgatag tcaactcctt ggttaatcac 5880 tttcttgttc gctggagttg ggagcatatg atggctagca catctctttt cttttcggac 5940 tgtgttgagc tggcagttgt tggggatgac aatttagtga gtgtgaagga ggtcgcagcc 6000 tttgatttca atttgaaaaa attgtctgct tttttagcag actttggttt caccctgaag 6060 gatggttccg acaagaataa ggagatcctc cctgagttca atccacctga aaagtgcgac 6120 tttctgaaga ggtgttttaa agcgcgtggg gatagatacc ttgctccact ctcctggtta 6180 tcactttctg agtcactcca ttgggttagg gagacaaata tgagtaatgc cgccgccaca 6240 caaaataatg tggagggatt tttaagagag ttgtttcatt acggggacaa aaatctgtat 6300 tgcaaatgga gaagggatct catagatctt tgttccaaaa atcgtgttcc ccacccaaca 6360 acctattcat ttgaggaatt ggagcgttct tggttgtctg ggaaatcagt tgcttccata 6420 tttgagaaag aggagccaga gatcattgtt atacgtgata gtgcttccga tgttgctccg 6480 ggtgttcata ttgtacctgt tcgacagtgc ctcaagtgga acgctggtga ggttcccaac 6540 gtcgtctggt gcggaccaaa ctgccccaat caacttagga atgctagcag ctgcttccag 6600 attcaagccc cccagggctc caagtatcct ctgcgcaata caacccgcaa tctcctgaag 6660 aaggttcatc agcgcgggga tgaagtttat tttactggat cactgaacca gtcgcttgtt 6720 cattgggtcg cagcttttta cgcctccatg taccgggaat cctttcatca ttctgcttat 6780 atgaaggcat attttgggga taatgatgca gggcttcttg aatcaaccat ggcagcaaaa 6840 ggctggtgag cccctcccct ttttcttagg catttctcct ggagaatatc cctggcccag 6900 acgggcacta atcaggcaag ccggtgaact tcgtgtgtca ctaccatgga tctacatggt 6960 tgttttactt ttctgtcata tcctgagggt caaaggtctt tgaaccgaag cccaagtatg 7020 gacaaaacat agtttagctt tttactttta gtatagggcc ttgttgtgtt agctcttcat 7080 tgagctgtac acttttgttg tgccaatctg ctttgtgtgt tttctttgga gccttctata 7140 tcttccttcg ttggatgtat gtagttgtta agctctttgc tttcttttgt gtttaaataa 7200 aggattaacc aggcatcctc ccaggtctta ccgggtttag ttctggcact tagataaagc 7260 tttaaaaaaa aaaaaaaaaa a 7281 <210> 2 <211> 3125 <212> DNA <213> Cnidium mottle virus <400> 2 ttgaaaagca atctgcgaac tttgttacga tttgttactt ccaactagct attgggctta 60 tccaatttct ctcgtttccg caatcactag caccgggctt atccggatct ctttttgttt 120 tctttgttac ctctgttctg tgcgcgcttt aattttcctt tgcgttcttt acagcagtca 180 caacgtttac attaagtaat ctcgcttccc cgatttgtca agttaggggt ttttgttttt 240 cttttttcgt ttcacagcaa acttttgctc ttttcctttt ctttctgctc ctctgctata 300 tagttcttcc ccctttgatt tctaacatgt cttcctttgg cagacgctca acacaagaga 360 tgccaaatgt tccttcatat tcccctgagg aaatggaagt tctgaaggct gccatcactg 420 aatatggaat tagttatgtt gatgtggtga gagcagctca aactgaaagt gggaagatgg 480 caattcttac tgcagtaagc acgaaccaac tggaaagtct agtagcaact actgctacta 540 gcaactcctt ggtgtgtttg cgtaagaagc ctgaagccac cgtccatgtt tctgagggtg 600 taagacgaca ggttctttgt gatgatgttc ctgcttttga ggaggatcag gcacaaagtg 660 gagaggctag agcctctttt tcctctgcaa acgcttctta tggacagcag gatagagtgg 720 ccctccccag cctacctccc caggagtgcc aatttcacaa taaagttggc tgcctctgta 780 ctcagcacac gagtggctcc gaagccagtg gtcatcatca tgaggagtta gttccagctt 840 cggagggagg tactgagggc cagttctttt ctccccaacc cattgcccat ccacaagact 900 cccgattcgt gggctctcat cccttctctt ttccaataaa ctccaatgta ggtaccgttg 960 tctatacttt acctattatg agaacctctc tgagggatac cgagtgggga agattttaca 1020 aaggctatcg ctacttaagg tgcaagccaa cggttcgtct gattggctcg gggcctatac 1080 aagcaaaggg tcttttgtgg ttgtgttatg atccctctga aactttgtcc aagtatccga 1140 gtagagag agctctagcc ttgcagggaa cttggtttat gccgggtagg catgattcag 1200 cttcgttaac tgttcaggaa ttagccaccc ccgcaggctt ttgtgacatg gatactgacg 1260 tcaatggtgc cttcaaggtg gttatcatca aggatctggc aaattttgat gtttctgact 1320 atgggatgga attatccttg tacttggagg tggaaaaaat aggacttgga agtgtggtga 1380 ctaatgaggc acttacaact ttttacccac tccgtcaagt agtcctggat tacgagctct 1440 caacaacaac ctcaaaaggg aaagcgcttg ttcttcctct caatcctttg cttcctgccc 1500 atgatgaggc tcaattttat ccgagttgtt cctcttctat cctggagaac cataggtact 1560 ggaagggtgt tctctcactg gaggttgtct tcaaccttcc cgcaatggca ggaggcattg 1620 ttgagcttgc attcgcatat aacacctatg ataatgtgga tggagatatt tataggcgct 1680 tcggctcctc agtggtggat ttaagagcac accgcatcct gcgtgctcgt gtgccattat 1740 ctggctatgg gggctatcta gctgggggtt ctggctcact ttttgaggtg aagccacaag 1800 taggctctgg agatactctc aaattggtgg tgttgtttac ggcacctctt catattagtg 1860 atacctcaaa gaaaggttct gtgctcgtta ggtaccttgg tcttgaagat ctggattacc 1920 tggaaccggc cacatccatt gggagactta atcctgaaac gactttagtc ccaaatgtcg 1980 ctgcttctgg tggaccaatt gtgaaggttg gtactgtcga gtgggaagag cctttcattt 2040 ctaaggtgcc tttgggctta cgtcaaaaga ccttccgtct aatgacaatt aataagtggg 2100 ctccctctgg tttcctttat tttcccgtta cgccttctgt gcatctgcca cgtacagctg 2160 gaaattttgt ggcggatctt gagcaacagt gccctttgat gcataggagt caggaaaatt 2220 gccagtggag gggcactctt aaataccatt tgacagctcg gctggaggga gccacggatc 2280 agctcattct tcctcatcgc tctcttactt tctctgctgt cttgttaagc aagatcttac 2340 cagctccgtg tttcatagat aatagtacct tcaagcctct gctccccttg ttgtctgtga 2400 aggacacctt ctctctggag caggatcatc cctttgtgga attcaccaca ccccctggta 2460 gatggaccaa tacacatttt ggtgccactg agcagtacac ctggcgcacc tgtccagtct 2520 gggttctcct ccaatttcca cccaagacaa tggcacagct ccacgtgagg gatgtttcgc 2580 tctgggttga gccggatatt gagtataggc atcccatggg cggtttccca ttgaccatcc 2640 ccgatccttc acctgcaccc aggtatttct ttgaaaacac tttcgttgtt ggatgagctc 2700 ttccttttcc ttaggcattt ctcccggaga atctccctgg cccagatggg caataatcag 2760 gcaagccggt gaactttgtg tgtcactacc atggatctac atggttgttt tacttttctg 2820 ccatatcctg agggtcaaag gtctttgaac cgaagcccaa gtatggacaa aaacataatt 2880 tagcttttta cttttagtat agggccttgt tgtgttagct cttcattgag ctgtacactt 2940 cttgttatgc caatctgctt tgtgtgtttt ctttggagcc ttctatctct tcctttgttg 3000 gatgtatgta gttgttaagc tctttgcttt cttttgtgtt taaataaagg attaaccagg 3060 catcctccca ggttttaccg ggtttagttc tggcacttag ataactcttt aaaaaaaaaa 3120 aaaaa 3125

Claims (3)

A Cnidium mottle virus gene consisting of the nucleotide sequence of SEQ. ID. NO: 1 and the nucleotide sequence of SEQ. A recombinant vector comprising the Cnidium mottle virus gene of claim 1. A host cell transformed with the recombinant vector of claim 2.