KR101035086B1 - Gene encoding ornithine decarboxylase antizyme protein from Spodoptera litura and uses thereof - Google Patents

Abstract

The present invention is a tobacco giant seminar ( Spodoptera) litura ) ODC Az (ornithine decarboxylase antizyme) protein, a gene encoding the protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, a host cell transformed with the recombinant vector and a host cell The present invention relates to a method of inactivating an ODC protein in an organism and a method of altering an ODC Az sequence to inactivate an ODC protein.

Tobacco germinants, ornithine decarboxylase antizyme, yeast, knockout, polyamines

Description

Gene encoding ornithine decarboxylase antizyme protein from Spodoptera litura and uses approximately

The present invention relates to an ODC Az protein coding gene derived from tobacco gelatin moth and its use.

Mankind is threatened even by food shortages caused by invasion of crops by harmful pests and pathogens. The easiest and most convenient way to control plant diseases is to spray pesticides, which are highly toxic, so that not only harmful pests and pathogens but also the surrounding ecosystem organisms can be solved. By killing, there have been harms such as destroying ecosystems and acidifying soil and water quality. As a result, the urgent need and necessity for the development of pollution-free biological pest control technology in consideration of the environment and conditions of plants as well as chemical control means such as spraying of pesticides are gradually increasing.

Polyamines, such as spermidine and spermine, are essential for the synthesis of proteins and nucleic acids, and require very high levels, especially in animal tissues such as testis that rapidly differentiate. Most biopolyamines are produced by putrescine from ornithine under the action of ornithine decarboxylase (ODC), and then various polyamines are synthesized from putrescine. ODC Az (antizyme) inhibits ODC intrinsic homodimer formation by forming heterodimer with ODC, while polyamine biosynthesis process is blocked as ODC-ODC Az heterodimer is degraded by proteosome.

The gene encoding ODC Az has a stop codon (TGA) in the frontal domain of its open reading frame (ORF), resulting in no translation of mRNA over its entire length, but only when spermidine rises above a certain level. Decoding proceeds with frame-shifting, which removes base T on ribosomes.

Thus, in insects, ODC Az is not detoxified and expressed unless the level of spermidine is excessively elevated, but if ORF is removed from the base T of the stop codon, the detoxification of ODC Az is not controlled. ODC activity is inhibited and polyamine deficiency can be induced to disturb normal occurrence.

In particular, the deficiency effect of polyamines may cause severe hindrance to spermatogenesis in testes where rapid cell division and differentiation progress, resulting in infertile individuals with impaired reproductive function.

Korean Patent Registration No. 10-0722001 discloses a topical composition for hair follicle delivery of an ornithine decarboxylase inhibitor, and US Patent No. 5811634 discloses a transgenic animal encoding ornithine decarboxylase.

The present invention has been made in accordance with the above requirements, and in the present invention, in order to develop a technique for controlling pests biologically by inhibiting ODC activity of a particular pest species using ODC Az, the main pests of vegetables are preferred. A cDNA encoding ODC was isolated from tobacco litterworm ( Spodoptera litura ) and transduced into ODC deficient yeast ( Saccharomyces cerevisiae ) to produce a yeast strain maintained by the action of insect ODCs. In addition, the cDNA encoding the ODC Az is isolated from the tobacco macrophages, and the structure of the ORF cDNA that can be deciphered through artificial point mutation is made, and then transduced into the yeast strain to functionalize the structurally modified insect ODC Az. The present invention was completed by confirming the expression.

In order to solve the above problems, the present invention provides an ODC Az (ornithine decarboxylase antizyme) protein derived from tobacco macropodia ( Spodoptera litura ).

The present invention also provides a gene encoding the protein.

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

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

The present invention also provides a method for inactivating ODC protein in a host cell by transforming the host cell with the recombinant vector.

The present invention also provides a method for altering the ODC Az sequence to inactivate the ODC protein.

The pest control technology of the new idea to be developed through the present invention has not yet been attempted globally, and if the project is successfully carried out, a biopesticide that can be used immediately on the agricultural site is produced and thus a great economic Valuable markets can be formed.

In addition, the immediate insecticidal effect may be slightly lower than that of conventional chemical insecticides, but in the long term, it may be more effective than conventional insecticides in that it may have a repetitive and chronic insecticidal effect remaining in the soil and environment. have.

In order to achieve the object of the present invention, the present invention is composed of the amino acid sequence of SEQ ID NO: 2, tobacco giant seminar ( Spodoptera litura ) provides ODC Az (ornithine decarboxylase antizyme) protein. SEQ ID NO: 2 is a sequence in which the 223rd base T is deleted from the nucleotide sequence of SEQ ID NO: 1 to perform frame-shifting to generate a complete ORF.

The range of the ODC Az protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from tobacco macropods and functional equivalents of the protein. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The present invention also provides a gene encoding the ODC Az protein. Genes of the invention include both genomic DNA and cDNA encoding ODC proteins. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. More preferably, the gene of the present invention is ODC AzM in which T, which is the 223th base of the nucleotide sequence of SEQ ID NO: 1, is deleted. Here, M means modification and means that the 223th base T is deleted.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a base sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with each of the nucleotide sequences of SEQ ID NO: 1. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the ODC Az gene according to the present invention. The ODC Az gene included in the recombinant vector is preferably ODC AzM in which T, which is the 223th base in the nucleotide sequence of SEQ ID NO: 1, is deleted. The recombinant vector is preferably a recombinant yeast expression vector.

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, a heterologous peptide, or a 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.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as a host cell, a promoter and an operator site of the E. coli tryptophan biosynthetic pathway, and a phage λ left promoter (pLλ promoter) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

The present invention also provides a host cell transformed with the recombinant vector of the present invention. The host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marsonsons and various Pseudomonas species Enterobacteria and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, yeast ( Saccharomyce) as a host cell cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), Hanahan method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) The vector can be injected into the host cell.

Preferably the host cell is yeast ( Saccharomyce cerevisiae ). In addition, preferably, the host cell may be one in which cell proliferation is inhibited and polyamine synthesis is reduced. As a result of overexpression of the ODC AzM gene of the present invention in yeast, the proliferation of yeast cells was inhibited and the synthesis of polyamines was reduced.

The present invention also provides a method of inactivating an ornithine decarboxylase (ODC) protein in a host cell comprising transforming the host cell with a recombinant vector comprising the ODC AzM gene of the present invention. ODC AzM overexpressed in yeast inhibits ODC intrinsic homodimer formation by forming heterodimer with ODC, while ODC-ODC Az heterodimer is decomposed by proteosome, blocking polyamine biosynthesis process. Therefore, host cell proliferation may be inhibited by the ODC protein inactivation, and polyamine synthesis may be reduced. The host cell is preferably yeast.

The present invention also provides

(a) isolating the ODC Az gene from the host cell;

(b) altering the ODC Az sequence to inactivate the ODC protein; And

(c) transforming the host cell with a recombinant vector comprising the altered ODC Az sequence to overexpress the altered ODC Az gene, thereby providing a method of inactivating an ornithine decarboxylase (ODC) protein in the host cell.

In the method of the present invention, the step of altering the ODC Az sequence to inactivate the ODC protein removes a stop codon occurring in the middle of the ORF so that the isolated ODC Az gene can be fully translated. It's a step. In this step, one base corresponding to the stop codon may be removed to induce frame-shifting, or one base corresponding to the stop codon may be replaced with another base to prevent termination so that a complete ORF may be generated.

In step (c), the host cell is transformed with a recombinant vector comprising the altered ODC Az sequence, wherein the host cell may be the same host cell from which the ODC Az gene is isolated, or the ODC Az gene is isolated. It may be a different host cell. That is, according to the purpose, it is possible to select the desired transformed host cell.

Altered ODC Az, overexpressed in host cells, can inactivate ODCs by forming heterodimers with ODCs, preventing the formation of homodimers inherent in ODCs.

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

1. Insect Sample

Tobacco Geopark , a major pest of various vegetable crops ( Spodoptera) litura ) larvae were fed in the growth chamber at 25 ± 1 ℃, 60% relative humidity, and photoperiod 16: 8 (L: D), and the testes were removed during the pupal stage and 1 ml per 100 mg. TRIzol reagent (Gibco BRL, USA) was added and stored at -70 ° C.

After crushing testis tissue in TRIzol reagent using micro-pestle, 0.2 ml of chloroform per 1 ml of TRIzol was added and shaken vigorously for 15 seconds, followed by centrifugation (12,000 xg, 15 min). Separated. Isopropanol was added to the supernatant, shaken, and centrifuged again (12,000 xg, 10 min) to obtain total RNA. 75% ethanol was added to the total RNA pellet, followed by vortexing to remove the supernatant by centrifugation (7,500 xg, 5 min), and the total RNA pellet was air dried at room temperature. Total RNA concentration was measured at a wavelength of 260 nm using a spectrophotometer (Spectronic Genesys 5) by dissolving the total RNA pellet with DEPC-H ₂ O.

After 200 ug of total RNA was reacted with 1 ug Oligo (dT) primer for 10 minutes at 70 ° C, it was allowed to stand on ice for 5 minutes, followed by 1st strand 5X buffer, RNasin ribonuclease inhibitor (40 units), sodium pyrophosphate (40 mM). ) And AMV reverse transcriptase (30 units) were added and reacted at 42 ° C. for 1 hour to synthesize first strand cDNA. To 20 ul of the synthesized first strand reactant, the second strand 2.5X buffer, DNA polymerase (23 units) and RNase H (0.8 unit) were added, and the total volume was adjusted to 100 ul using distilled water and reacted at 14 ° C. for 2 hours. The reaction solution was again heated at 70 ° C. for 10 minutes, and then added to T4 DNA polymerase (2 units) while standing on ice for 10 minutes at 37 ° C., followed by addition of 200 mM EDTA (10 ul). The reaction was terminated. To recover the cDNA, the same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the reaction solution, followed by vortexing, followed by centrifugation (15,000 rpm, 2 min). Sodium acetate (pH 5.2) and 2.5 volumes of 100% EtOH (-20 ° C.) were added and allowed to stand at −70 ° C. for 30 minutes. Subsequently, the cDNA pellet precipitated through centrifugation (15,000 rpm, 10 min) was washed with 70% EtOH (-20 ° C) and finally dissolved in an appropriate amount of DW. The concentration of cDNA was measured at a spectrophotometer wavelength of 260 nm. It was.

2. Tobacco Seminar Room ODC Coding cDNA Separation of

2-1. Partial domain fabrication of ODC-coded cDNA

5 ′ gene specific primers (5′-GGGCACTGGCTTCGATTGCGC-3 ′; SEQ ID NO: 3) and 3′degenerate to hybridize target sequences on a cDNA template based on previously reported base sequences of insect ODCs to amplify partial domains of ODCs A primer (5'-YYAYTTSAGSGCRCAYTCSAC-3 '; SEQ ID NO: 4) was prepared.

PCR was performed using MJ Resaech, Inc's Model PTC-100 Programmable Thermal Cycler under Table 1, with 0.25 mM dNTPs, 0.2 mM MgCl ₂ , 5 'and 3' primer pools of 10 uM and 20 uM, respectively, and a 100 ng cDNA template. The reaction volume was 50 ul, which was used. As a template, cDNA synthesized from total RNA extracted from testis tissue was used. The PCR reaction was initiated by preincubation at 95 ° C. for 5 minutes and then adding 0.4 ul Taq polymerase (TaKaRa Ex-Taq) at 80 ° C. The amplified cDNA was identified and purified by electrophoresis on agarose gel (FIG. 1), and then inserted into a plasmid pCR 2.1 vector using a TA cloning kit (Invitrogen) to analyze its nucleotide sequence. The tissue was confirmed to be ODC cDNA ( Slit ODC).

Table 1. PCR thermal cyclization profiles for amplifying partial domains of ODC-encoded cDNA in tobacco macromoth

Preheating 95 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle
Reaction
Denaturation 95 ℃ for 1min
39 cycles
Annealing 58 ℃ for 1min Extension 72 ℃ for 2min Last extension 72 ℃ for 5min 1 cycle

2-2. 5 'and 3' end fabrication of ODC-coded cDNA

Genetic specific RACE primers were constructed to amplify the 5 'and 3' ends of the ORF, based on the nucleotide sequence of the cDNA partial domain identified by the structural analysis encoding ODC Az (5 'primer, 5'-TGTCATTCCACGTGGGTAGCG). -3 '(SEQ ID NO: 5); 3' primer, 5'-GGGGAAGTGCTCCTCCAAAGC-3 '(SEQ ID NO: 6)).

RACE PCR was performed using a Hot Start Taq DNA Polymerase (Takara) with a cDNA with an Ap1 adapter made using the Marathon cDNA Amplification Kit (Clontech) (0.25 mM dNTPs, 0.2 mM MgCl ₂ , 10 each). uM 5 ′ and 3 ′ primers, 100 ng cDNA) (Table 2). PCR products of sufficient length that meet both ends were identified and purified by electrophoresis (FIG. 2) and inserted into the pCR2.1 vector and subjected to E. coli The nucleotide sequence was analyzed by cloning.

Table 2. Thermal cyclization profiles in RACE PCR

Preheating 94 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle Preheating 94 ℃ for 30sec 1 cycle

Reaction


Denaturation 94 ℃ for 30sec 5 cycles Annealing 67 ℃ for 4min Denaturation 94 ℃ for 30sec 5 cycles Annealing 65 ℃ for 4min Denaturation 94 ℃ for 20sec 35 cycles Annealing 62 ℃ for 4min Last extension 72 ℃ for 5min 1 cycle

2-3. Full length of ODC-coded cDNA-construction of ORF cDNA

Gene specificity corresponding to the initiation and termination sites of each ORF to amplify full-length ORF cDNAs based on 5 'and 3' terminal sequences containing start codons (ATG) and stop codons (TGA) identified by RACE PCR Red primers were prepared (5 'primer, 5'-ATGAAGGTCGTGGAA-3' (SEQ ID NO: 7); 3 'primer, 5'-TCACTTGAGCGCGCACTC-3' (SEQ ID NO: 8)), using them under the conditions of Table 3 Was carried out. The size of the amplified full-length ORF cDNAs was confirmed on agarose gel (FIG. 3), inserted into pCR2.1 vector and E. coli , respectively. The nucleotide sequence was analyzed by cloning.

Table 3. PCR thermal cyclization profiles for amplification of full-length ODC ORF cDNA

Preheating 95 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle
Reaction
Denaturation 95 ℃ for 1min
39 cycles
Annealing 54 ℃ for 1min Extension 72 ℃ for 2min Last extension 72 ℃ for 5min 1 cycle

2-4. Slit with Yeast Expression Vector Cloning of ODC

Slit inserted in the pCR vector The ODC cDNA was digested with EcoRI to isolate 1.3 kb fragments, and then ligated to p413GAL1 vector digested with EcoRI (p413GAL- Slit ODC, FIG. 4). The Slit ODC gene is transcribed in a medium containing galactose by the GAL1 promoter of the P413GAL1 vector, and transcription is terminated by the CYC1 terminator.

3. Tobacco Seminar Room ODC Az  Coding cDNA Separation of

3-1. Partial domain fabrication of ODC Az coding cDNA

In order to construct about 430 bp conserved core domain of ODC Az, primers were constructed to hybridize target sequences from cDNA based on previously reported nucleotide sequences of ODC Az insect species (5 ′ primer, 5′-CTGTGGTGGTCCTGATGTTCC-3 ′ (SEQ ID NO: 6). Number 9); 3 'primer, 5'-CTCTTCGGCGAAATCCAGAG-3' (SEQ ID NO: 10)). PCR was carried out in a reaction volume of 50 ul consisting of 0.25 mM dNTPs, 0.2 mM MgCl ₂ , 5 'and 3' primer pools of 4 uM each, and a 100 ng cDNA template under the conditions shown in Table 4. The cDNA synthesized from total RNA was used, and preincubated at 95 ° C. for 5 minutes, and then started by adding 0.4 ul Taq polymerase (TaKaRa Ex-Taq) at 80 ° C. After confirming the amplified cDNA by electrophoresis on agarose gel (Fig. 5), it was confirmed that the ODC Az cDNA ( Slit ODC Az) of the testicular tissue of tobacco macrogonist by the same method as described above.

Table 4. PCR thermal cyclization profiles for amplifying the 340 bp core domain of ODC Az-encoded cDNAs in tobacco litter

Preheating 95 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle
Reaction
Denaturation 95 ℃ for 1min
39 cycles
Annealing 62 ℃ for 1min Extension 72 ℃ for 2min Last extension 72 ℃ for 5min 1 cycle

3-2. Fabrication of 5 ′ and 3 ′ ends of ODC Az-coded cDNA

Gene-specific RACE primers were prepared based on the nucleotide sequence of the core domain cDNA identified as ODC Az by structural analysis (5 'primer, 5'-TCACCGAGTCCCAGTCTCCCA-3' (SEQ ID NO: 11); 3 'primer;5'-GCTTCTCTGCGTTGCTGGTGG-3'(SEQ ID NO: 12), RACE PCR was performed in the same manner as described above (0.25 mM dNTPs, 0.2 mM MgCl ₂ , 10 uM 5' and 3 'primers each, 50 ng cDNA) 5). PCR products of sufficient length that meet both ends of the ORF were confirmed by electrophoresis (FIG. 6), and then inserted into the pCR2.1 vector in the same manner as above and then E. coli The nucleotide sequence was analyzed by cloning.

Table 5. Thermal cyclization profiles in RACE PCR

Preheating 94 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle Preheating 94 ℃ for 30sec 1 cycle

Reaction


Denaturation 94 ℃ for 30sec 5 cycles
Annealing 68 ℃ for 4min Denaturation 94 ℃ for 30sec 5 cycles
Annealing 66 ℃ for 4min Denaturation 94 ℃ for 20sec 35 cycles
Annealing 64 ℃ for 4min Last extension 72 ℃ for 5min 1 cycle

3-3. Full-length ORF cDNA Fabrication of ODC Az-coded cDNA

Bases of the start and end sites of each ORF to amplify the full-length ORF cDNA based on sequences of 5 'and 3' ends that normally contain start codons (ATG) and stop codons (TAA) identified by RACE PCR making a specific gene was added to the BamHI and SacI sequences 5 'and 3' ends of the respective sequence corresponding to the sequence primer pair (5 'primer, 5'-GGG GGATCC ATG ACGAAGTTAATACAACAA- 3' ( SEQ ID NO: 13) PCR was performed under the conditions of 3 ′ primer, 5′-GGG GAGCTC TTA CTGCATATTGTAGTGCAG-3 ′ (SEQ ID NO: 14), and Table 6. The size of the full-length ORF cDNAs amplified by PCR was confirmed on an agarose gel (FIG. 7), inserted into a pCR 2.1 vector, and E. coli The nucleotide sequence was analyzed by cloning.

Table 6. PCR thermal cyclization profiles for amplification of full-length ODC Az ORF cDNA

Preheating 95 ℃ for 5min 1 cycle Annealing 80 ℃ for 5min 1 cycle
Reaction
Denaturation 95 ℃ for 1min
39 cycles
Annealing 62 ℃ for 1min Extension 72 ℃ for 2min Last extension 72 ℃ for 5min 1 cycle

3-4. Slit with Yeast Expression Vector Cloning of ODC Az

Slit inserted in the pCR vector ODC Az cDNA was digested with BamHI and Sal1 to separate fragments, and then ligated to pG1 vector digested with the same restriction enzymes (pG1- Slit ODC Az).

4. Tobacco Seminar Room ODC Az cDNA Variation of Modification )

Gene specific for T deletion to remove T (223-T), base 223, from the stop codon (TGA) at bases 223-225 of ODC Az ORF cDNA using site-directed mutagenesis kit (Stratagene) Red primers (point mutated primers # 1, 5'-CCTCTGTGGTGGTCCGATGTTCCGGCTCAG-3 '(SEQ ID NO: 15); point mutated primers # 2, 5'-GTCACGCGGAACATCGGACCACCACAGAGG-3' (SEQ ID NO: 16)) were prepared. PCR was carried out using a reaction volume of 50 ul consisting of 5 ul 10x buffer, 1 ul dNTP mix, 3 ul quicksolution, 125 ng of primer # 1 and # 2 and 50 ng dsDNA template under the conditions of Table 7. The structure-reformed ODC Az cDNA ( Slit ODC AzM) derived from the removed tobacco macromolecular testis tissue was amplified.

Table 7. PCR thermal cyclization profiles for amplifying point mutated ODC Az (ODC AzM) cDNA in tobacco litter

Preheating 95 ℃ for 5min 1 cycle
Reaction
Denaturation 95 ℃ for 50sec
18 cycles
Annealing 60 ℃ for 50sec Extension 68 ℃ for 5min Last extension 68 ℃ for 7min 1 cycle

5. ODC  And ODC Az  Construction of Deficiency Yeast Strains

5-1. Yeast strain

W303-1A ( MATa Saccharomyces cerevisiae strains of ade2 , ura3 , his3 , trp1 , leu2 , can1 were used, and YPD (yeast extract-peptone-dextrose), or synthetic complete minimal medium (0.67% yeast nitrogen base w / o aa, 2% glucose and amino acids, except those required for selection).

5-2. Knockout of the ODC Gene ( SPE1 )

ODC gene ( SPE1 ) was amplified by PCR reaction from chromosomal DNA of yeast W303-1A using SPE1-5 and SPE1-3 primers, and the amplified 1.4 kb of DNA was digested with BamHI and XhoI to obtain a pBluescript SK (+) vector. Cloned into (p SPE1 ). The p SPE1 plasmid was digested with HincII and HindIII, eluted from agarose gel, filled-in with Klenow fragments, and ligated to the URA3 gene digested with PvuII (pSPE1 :: URA3) (FIG. 8).

The fragment with the URA3 gene was amplified with SPE1-5 and SPE1-3 primers to isolate a 2.5 kb PCR fragment, which was then transformed into a W303-1A or W303-1A OAZ1 :: Hy strain and transformed into a SPE1 gene on a chromosome. The homologous recombination occurred to knock out the SPE1 gene on the chromosome (FIG. 9). DNA was isolated from the transformants and PCR with SPE1-5 and SPE1-3 primers to confirm the presence of 2.5 kb knocked out genes instead of 1.4 kb SPE1 genes (FIG. 10).

5-3. Knockout of the ODC Az Gene ( OAZ1 )

Yeast ODC antizyme 1 gene ( OAZ1 ) was amplified by PCR reaction using AZ1-5 and AZ1-3 primers from chromosomal DNA of strain W303-1A. Amplified 1.2 kb of DNA was cloned into pHNT-T vector, cleaved with BamHI and XhoI and cloned back into pBluescript SK (+) vector (pBAZ1). The pBAZ1 plasmid was digested with EcoRV and HindIII, eluted from agarose gel, and then digested and extracted from the pG1-Hy plasmid with the same restriction enzymes from the hygromycin resistance gene, including the glycerate phosphate dehydrogenase promoter and the phosphoglycerate kinase terminator. PBAZ1 DNA was ligated (pBAZ1 :: Hy) (FIG. 11).

Using AZ1-5 and AZ1-3 primers, amplified 3.5 kb PCR fragment containing hygromycin resistance gene ( Hy ) was transformed into W303-1A strain and homologous recombination with OAZ1 gene on chromosome. By knocking out the OAZ1 gene on the chromosome (Fig. 12), DNA was isolated from the transformant and PCR with the AZ1-5 and AZ1-3 primers to obtain a 3.5 kb knocked out gene with Hy inserted in place of the 1.2 kb OAZ1 gene. It was confirmed (FIG. 13).

6. Transformation of Yeast

Overnight culture inoculated with a yeast strain W303-1A in YPD medium, and (10 ⁵ to 10 ⁶ cells / ml), 30 ℃, incubated at 180 rpm for about 5 hours, the number of cells by centrifugation when the increase of about 10 times (5,000 rpm , 10 min), the cells were collected, washed with distilled water, suspended in 1 mL of 0.1 M LiOAc solution, then centrifuged at 12,000 rpm for 30 seconds, and then suspended in 450 μl of the same solution. 50 μl of the cell solution was placed in a centrifuge tube and centrifuged in the cell pellet obtained in the transfection mixture in order (240 μl 50% PEG3350, 36 μl 1 M LiOAc, 5 μl 10 mg / ml ss DNA, 60 μl DW, 10 μl plasmid DNA) was added and vortexed. It was reacted at 30 ° C. for 30 minutes and subjected to heat shock at 42 ° C. for 25 minutes, and then cell pellets were recovered by centrifugation (12,000 rpm, 30 sec). The cells resuspended in distilled water were plated on a synthetic minimal medium plate and incubated at 30 ° C. for 2 days.

7. Change of structure Tobacco Seminar Room ODC AZ  ( Slit ODC AzM Expression check

7-1. Culture and Growth Rate Analysis of Transgenic Yeast

To identify the gene expression patterns in yeast cells transformed by ODC and ODC Az-coding cDNAs of tobacco castor moth, the selection medium (+ Ura / glucose, -Ura / glucose, -Ura / Galactose and -Leu -Ade / galactose) were inoculated with 10 ⁴ cells / ml each and shaken incubated for 24 to 72 hours at 30 ° C. and 180 rpm. After diluting 1 ml of each cell culture solution 50 times, a microscope (x) was used with a hemacytometer. The cell number was counted under 400).

7-2. Cell polyamine content analysis

Intracellular polyamine levels of yeast were dissolved in 2 mg of 20% perchloric acid in 10 mg of cultured yeast cells for 1 to 2 minutes, and the supernatant obtained by centrifugation (3,000 rpm) was dissolved in Bio-Rad AG 50W-X4 anion exchange resin ( Loaded into a 10 ml column of Kontes Econoflex (Biorad) filled with H ⁺ form, 200-400 mesh), washed sequentially with 0.7 M NaCl (pH 8.0), DW, 1 N HCl in 0.1 M sodium phosphate, and then Elution with HCl produced a polyamine sample.

25 ul of OPT (o-phthaldehyde-mercaptoethanol) reagent was added to 200 ul of polyamine sample and reacted for 1 minute, and 100 ul was subjected to high performance liquid chromatography (HPLC, Waters system 600 controller, 474 Scanning fluorescence detector-340/455 The levels of putrescine, spermidine and spermine were measured by injection into the nm excitation / emission filter. HPLC analysis was performed using an Altex Ultrasphere-ODS column (5 μm, 4.6 mm x 15 cm) equipped with a C ₁₈ Brownlee lab guard column, 0.1 M with methanol (solvent A) and 0.05 M DCMA (N, N-dimethylcyclohexylamine) added. Gradient program (0-2 min, 80% B at 20% A; 20% B linear at 2-10 min, 80% A) at 1 ml / min with phosphate buffer (pH 4.0) (solvent B) as eluent gradient; 2.3 min hold, 12.3-12.5 min linear to initial condition).

Example  1: yeast ODC  gene ( SPE1 Knockout effect

1. Proliferation Rate

The growth rate of the ODC deficient strain ( SPE1 :: Ura3 ) knocked out the ODC gene ( SPE1 ) in the W303-1A strain was compared with that of the wild type strain (Table 8, FIG. 14). At 53% and 79% of the wild type strains, respectively, it was found that the inhibition of ODC expression due to knockout of SPE1 inhibited the proliferation of cells.

Table 8. Cell Numbers in Liquid Culture of Wild-Type and ODC Deficient Yeast Strains

Strain 24 hrs 48 hrs Wild type 2.20 × 10 ⁸ cells / ml 2.54 × 10 ⁸ cells / ml ODC deficiency ( SPE1 :: Ura3 ) 1.16 × 10 ⁸ cells / ml 2.01 × 10 ⁸ cells / ml

2. Polyamine Level

Cell polyamine levels of the ODC deficient strain ( SPE1 :: Ura3 ) knocking out the ODC gene ( SPE1 ) in the W303-1A strain were compared with the wild type strain at 48 hours of culture (Table 9, FIG. 15 and FIG. 16), SPE1 knockout strain. In putrescine is reduced by 56%, spermidine 28%, spermine 29%, respectively, the effect of reducing polyamine synthesis due to the lack of ODC was clearly observed.

Table 9. Polyamine Content of Wild-type and ODC Deficient Yeast Strains

Strain
Content * (pg / mg cells) Putrescine Spermidine Spermine Wild type 8.60 62.49 215.43 ODC Infection ( SPE1 :: Ura3 ) 4.89 17.49 62.33

* Measured at 48 hours of incubation

Example  2: In yeast Tobacco Seminar Room ODC cDNA  Expression effect

1. Proliferation Rate

ODC ODC gene deficient strain was a knockout (SPE1) (SPE1 :: Ura3) the tobacco giant seminar room into the vector p413Gal ODC (Slit ODC) Comparison of the growth rate of the strain transformed with cDNA ( SEP1 :: Ura3 / p413Gal- Slit ODC) and the strain transformed with the vector without Slit ODC cDNA ( SEP1 :: Ura3 / p413Gal) (Table 10 17) The number of cells of the strain into which the Slit ODC was introduced increased to about 1.8- and 1.6-fold levels of the control at 48 and 72 hours of culture, respectively, so that the ODCs of tobacco macromolecules transduced in yeast were subjected to SPE1 knockout. It has been found to successfully complement the lack of yeast ODC.

Table 10. Cell Number in Liquid Culture of Yeast Strains or Untransformed Yeast Strains Transformed with Slit ODC cDNA

Strain 48 hrs 72 hrs SEP1 :: Ura3 / p413Gal- Slit ODC ^A 6.44 × 10 ⁷ cells / ml 9.44 × 10 ⁷ cells / ml SEP1 :: Ura3 / p413Gal ^B 3.63 × 10 ⁷ cells / ml 5.81 × 10 ⁷ cells / ml

A, transformed with Slit ODC cDNA of p413Gal vector;

B, only transformed with p413Gal vector

2. Polyamine Level

Slit Cell polyamines of the strain ( SEP1 :: Ura3 / p413Gal- Slit ODC) introduced with ODC cDNA were analyzed at 72 hours of culture (Table 11, FIG. 18 and FIG. 19), and the control group ( SEP1 :: Ura3 / p413Gal). Compared to putrescine 44.9 times, spermidine 4.2 times, spermine 38.8 times higher, the complementation effect on the yeast ODC gene ( SPE1 ) of the tobacco gelatin moth ODC cDNA was clearly confirmed.

Table 11.Polyamine Content of Yeast Strains or Untransformed Yeast Strains Transformed with Slit ODC cDNA

Strain
Content * (pg / mg cells) Putrescine Spermidine Spermine SEP1 :: Ura3 / p413Gal- Slit ODC ^A 275.25 172.74 326.92 SEP1 :: Ura3 / p413Gal ^B 6.13 41.38 8.43

A, transformed with Slit ODC cDNA of p413Gal vector;

B, only transformed with p413Gal vector;

* Measured at 72 hours of incubation

Example  3: restructuring Slit ODC Az  ( ODC AzM )of Slit ODC Inhibitory effect on

1. Proliferation Rate

Tobacco Seminar ODC ( Slit ODC) Strain transformed into yeast strain ( SEP1 :: Ura3 / p413Gal- Slit ODC) complemented with cDNA with PG1 vector incorporating ODC Az ( Slit ODC AzM) cDNA ( SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC AzM) and a strain transformed with a PG1 vector inserted with an unmodified structure of tobacco macrosaeng ODC Az ( Slit ODC Az) cDNA ( SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC Az) proliferation rate (Table 12, Fig. 20), the number of cells of the strain in which the structural change Slit ODC Az cDNA was introduced, the unique Slit ODC Az was introduced at 24 hours and 48 hours, respectively Reducing the levels of 43% and 56% of the control group, it was confirmed that the structural altered ODC Az cDNA expression of tobacco moth moths has an inhibitory effect on the tobacco moth ODC.

Table 12. Cell number in liquid culture of yeast strains transformed with modified or original Slit ODC Az cDNA

Strain 24 hrs 48 hrs SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC AzM ^A 1.87 × 10 ⁷ cells / ml 8.37 × 10 ⁷ cells / ml SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC Az ^B 4.31 × 10 ⁷ cells / ml 1.48 × 10 ⁸ cells / ml

A, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC AzM cDNA of pG1 vector;

B, Slit ODC cDNA of p413Gal vector and Slit ODC Az cDNA of pG1 vector

2. Polyamine Level

Structural Changes of Tobacco Semicoated Moths Cell polyamines of strains ( SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC AzM) incorporating ODC Az ( Slit ODC AzM) cDNA were analyzed at 48 hours of culture (Table 11, 18 and 19), compared to the control ( SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC Az) in which the structure of the tobacco-coated semi-ovary-specific ODC Az ( Slit ODC Az) cDNA was not changed. Trecine decreased to 40.1%, spermidine 64.1%, and spermin 58.6%. This suggests that the alteration of the removal of base T from the termination codons present in the ODC Az ORF associated with frame-shifting in polyamine metabolism allows ODC Az to be expressed at all times and that the expressed ODC Az effectively inhibits ODC activity. To prove that.

Table 13. Polyamine Content of Yeast Strains Modified or Modified with Original Slit ODC Az cDNA

Strain
Content * (pg / mg cells) Putrescine Spermidine Spermine SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC AzM ^A 7.71 52.53 10.84 SPE1 :: Ura3 / p413Gal- Slit ODC / pG1-ODC Az ^B 19.21 82.02 18.50

A, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC AzM cDNA of pG1 vector;

B, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC Az cDNA of pG1 vector;

* Measured at 48 hours of incubation

Degree Figure 1 shows the PCR product encoding the core domain (1,110 bp) of ODC amplified from testicular cDNA of tobacco macromolecular moth.

1, partial domain fragment;

M, size marker

Figure 2 shows the RACE PCR product of ODC cDNA in the testis of tobacco castor moth.

3 ', insertion of 3' end into pCR2.1 vector;

5 ', insertion of 5' end into pCR2.1 vector;

M, size marker

Figure 3 is alit Slit amplified from testicular cDNA of tobacco giant Show the ORF cDNA of the ODC.

1, full length ORF cDNA;

M, size marker

4 shows the recombinant yeast expression vector p413GAL- Slit. It is a schematic diagram of ODC.

Degree 5 shows a PCR product encoding the core domain (1,110 bp) of ODC Az amplified from testicular cDNA of tobacco macromolecular moth.

1, partial domain fragment;

M, size marker

Figure 6 shows the RACE PCR product of ODC Az cDNA in the testis of tobacco weed.

3 ', insertion of 3' end in pCR2.1 vector;

5 ', insertion of 5' end in pCR2.1 vector;

M, size marker

Figure 7 Slit amplified from testicular cDNA of tobacco macromoth The ORF cDNA of ODC Az is shown.

1, full length ORF cDNA;

M, size marker

8 is yeast ( Saccharomyces) cerevisiae ) shows the insertion of URA3 into the ODC gene ( SPE1 ).

9 is yeast ( Saccharomyces) cerevisiae ) shows knockout of the ODC gene ( SPE1 ).

10 is yeast ( Saccharomyces) cerevisiae ) is the result of identification of knocked out ODC gene ( SPE1 ) with URA3 insert.

11 is yeast ( Saccharomyces) cerevisiae ) shows the insertion of Hy into the ODC Az gene ( OAZ1 ).

12 is yeast ( Saccharomyces) cerevisiae ) shows knockout of the ODC Az gene ( OAZ1 ).

13 is yeast ( Saccharomyces) cerevisiae ) knocked out ODC Az gene ( OAZ1 ) with Hy insert.

14 is a comparison of the growth rates between wild type (A) and ODC deficient (B) strains.

15 is an HPLC chromatogram of polyamines from wild type (A) and ODC deficient (B) yeast strains.

PUT, putrescine; SPD, spermidine; SPM, spermine

16 is a comparison of cellular polyamine content between wild type (A) and ODC deficient (B) yeast strains.

PUT, putrescine; SPD, spermidine; SPM, spermine

17 is a comparison of cell numbers between strains (A) or strains (B) transformed with Slit ODC cDNA.

A, transformed with Slit ODC cDNA of p413Gal vector;

B, only transformed with p413Gal vector

FIG. 18 is an HPLC chromatogram of polyamines of yeast strain (A) transformed with Slit ODC cDNA of p413Gal vector and only yeast strain (B) transformed with p413Gal vector.

PUT, putrescine; SPD, spermidine; SPM, spermine

19 is a comparison of cellular polyamine content between yeast strains (A) or untransformed yeast strains (B) transformed with Slit ODC cDNA.

A, transformed with Slit ODC cDNA of p413Gal vector;

B, only transformed with p413Gal vector

PUT, putrescine; SPD, spermidine; SPM, spermine

20 is a comparison of cell numbers of cultured yeast strains transformed with modified (A) or original (B) Slit ODC Az cDNA.

A, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC AzM cDNA of pG1 vector;

B, Slit ODC cDNA of p413Gal vector and Slit ODC Az cDNA of pG1 vector

21 is an HPLC chromatogram of polyamines of yeast strains transformed with modified (A) or original (B) Slit ODC Az cDNA.

A, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC AzM cDNA of pG1 vector;

B, Slit ODC cDNA of p413Gal vector and Slit ODC Az cDNA of pG1 vector

PUT, putrescine; SPD, spermidine; SPM, spermine

22 is a comparison of cellular polyamine content between yeast strains transformed with modified (A) and original (B) Slit ODC Az cDNA.

A, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC AzM cDNA of pG1 vector;

B, transformed with Slit ODC cDNA of p413Gal vector and Slit ODC Az cDNA of pG1 vector;

PUT, putrescine; SPD, spermidine; SPM, spermine

<110> Hannam University Institute for Industry-Academia Cooperation          Nexgen Biotechnologies, Inc. <120> Gene encoding ornithine decarboxylase antizyme protein from          Spodoptera litura and uses <130> PN09142 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 814 <212> DNA <213> Spodoptera litura <400> 1 atgacgaagt taatacaaca attaaatttt agtagtagta ttagtagtaa ttataataat 60 tactacgacg tcgctggctt ggaaccaccc gctgttgaag agaagaatta ctatggcgac 120 agcgcgtctt tgagcgcggc cggcagcaag cgctcggctt tgtcggcttc cgacgccgag 180 tgtttctcgc tgtgcctggg tgccgggcct ctgtggtggt cctgatgttc cggctcacgg 240 ctcggcaccg cccggagggg tgaatggcgg ggccgcgagc cccgctaccc catctacacc 300 cacccacgac gacaacaacc gtgacttgtt gagcgcccta ctatgggcaa gcgcgagttc 360 cctcgcgtcc agcgcggagt ctctcgtcac cgagtcccag tctcccacgc atgcgcaccc 420 agcactccac cagcaacgca gagaagcggt tataagcaag attctggaac gcaaggacag 480 gcagccggta aaaatcgact ttaagatctt ccttactgag aacacggtga cccgctggga 540 agctgtggtc caaggtggca ctatctacct gcgtatgccg ggagtccttc agtccggcag 600 taaggagagc ttcatgctac ttctggactt cgccgaggaa aggctggggt gtaacaactg 660 catcatctgc gtcctgaaga gccgaccgga ccgcgccacc attctccgca ctttcatgtt 720 catgggattc cagttactcc caccaaactc ccctcttatg ccgcaagaaa tcaccaaccc 780 ggagtacata ttcctgcact acaatatgca gtaa 814 <210> 2 <211> 270 <212> PRT <213> Spodoptera litura <400> 2 Met Thr Lys Leu Ile Gln Gln Leu Asn Phe Ser Ser Ser Ile Ser Ser   1 5 10 15 Asn Tyr Asn Asn Tyr Tyr Asp Val Ala Gly Leu Glu Pro Pro Ala Val              20 25 30 Glu Glu Lys Asn Tyr Tyr Gly Asp Ser Ala Ser Leu Ser Ala Ala Gly          35 40 45 Ser Lys Arg Ser Ala Leu Ser Ala Ser Asp Ala Glu Cys Phe Ser Leu      50 55 60 Cys Leu Gly Ala Gly Pro Leu Trp Trp Ser Asp Val Pro Ala His Gly  65 70 75 80 Ser Ala Pro Pro Gly Gly Val Asn Gly Gly Ala Ala Ser Pro Ala Thr                  85 90 95 Pro Ser Thr Pro Thr His Asp Asp Asn Asn Arg Asp Leu Leu Ser Ala             100 105 110 Leu Leu Trp Ala Ser Ala Ser Ser Leu Ala Ser Ser Ala Glu Ser Leu         115 120 125 Val Thr Glu Ser Gln Ser Pro Thr His Ala His Pro Ala Leu His Gln     130 135 140 Gln Arg Arg Glu Ala Val Ile Ser Lys Ile Leu Glu Arg Lys Asp Arg 145 150 155 160 Gln Pro Val Lys Ile Asp Phe Lys Ile Phe Leu Thr Glu Asn Thr Val                 165 170 175 Thr Arg Trp Glu Ala Val Val Gln Gly Gly Thr Ile Tyr Leu Arg Met             180 185 190 Pro Gly Val Leu Gln Ser Gly Ser Lys Glu Ser Phe Met Leu Leu Leu         195 200 205 Asp Phe Ala Glu Glu Arg Leu Gly Cys Asn Asn Cys Ile Ile Cys Val     210 215 220 Leu Lys Ser Arg Pro Asp Arg Ala Thr Ile Leu Arg Thr Phe Met Phe 225 230 235 240 Met Gly Phe Gln Leu Leu Pro Pro Asn Ser Pro Leu Met Pro Gln Glu                 245 250 255 Ile Thr Asn Pro Glu Tyr Ile Phe Leu His Tyr Asn Met Gln             260 265 270 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> 5 'gene-specific primer <400> 3 gggcactggc ttcgattgcg c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3 'degenerate primer <400> 4 yyayttsags gcrcaytcsa c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer <400> 5 tgtcattcca cgtgggtagc g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer <400> 6 ggggaagtgc tcctccaaag c 21 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer <400> 7 atgaaggtcg tggaa 15 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer <400> 8 tcacttgagc gcgcactc 18 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer <400> 9 ctgtggtggt cctgatgttc c 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer <400> 10 ctcttcggcg aaatccagag 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer <400> 11 tcaccgagtc ccagtctccc a 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer <400> 12 gcttctctgc gttgctggtg g 21 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer <400> 13 gggggatcca tgacgaagtt aatacaacaa 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer <400> 14 ggggagctct tactgcatat tgtagtgcag 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> point mutated primer # 1 <400> 15 cctctgtggt ggtccgatgt tccggctcag 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> point mutated primer # 2 <400> 16 gtcacgcgga acatcggacc accacagagg 30  

Claims (12)

An ODC Az (ornithine decarboxylase antizyme) protein from Spodoptera litura , consisting of the amino acid sequence of SEQ ID NO: 2. The gene encoding the ODC Az protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. The gene according to claim 3, wherein the 223nd base T in the nucleotide sequence of the gene SEQ ID NO: 1 is deleted ODC AzM. Recombinant vector comprising the gene of claim 4. A host cell transformed with the recombinant vector of claim 5. 7. The host cell of claim 6, wherein cell proliferation is inhibited and polyamine synthesis is reduced. The host cell according to claim 6, which is a yeast. A method of inactivating an ornithine decarboxylase (ODC) protein in a host cell comprising transforming the host cell with the recombinant vector of claim 5 to overexpress the ODC AzM gene. 10. The method of claim 9, wherein the ODC protein inactivation inhibits host cell proliferation and reduces polyamine synthesis. 10. The method of claim 9, wherein said host cell is yeast. Isolating the ODC Az gene from the host cell; Altering the ODC Az sequence to inactivate the ODC protein; And A method of inactivating an ornithine decarboxylase (ODC) protein in a host cell, comprising overexpressing the altered ODC Az gene by transforming the host cell with a recombinant vector comprising the altered ODC Az sequence.

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