KR20130104629A - Argonaute 4 of brassica rapa ssp. pekinensis and a gene encoding the same - Google Patents

Abstract

PURPOSE: Brassica rapa ssp. Pekinensis-derived argonaute 4 protein and a gene encoding the same are provided to construct RNAivector using isolated argonaute 4 gene and to produce transgenic Brassica rapa ssp. Pekinensis. CONSTITUTION: A primer gene which encodes Brassica rapa ssp. Pekinensis-derived argonaute 4 protein has base sequences of sequence numbers 1 and 2. The gene has a base sequence of sequence number 3. Argonaute 4 protein has an amino acid sequence of sequence number 4. A recombinant plasmid is prepared by cloning argonaute 4 gene in pGEM-T easy vector.

Description

Arganote 4 protein derived from cabbage and its coding gene {Argonaute 4 of Brassica rapa ssp. pekinensis and a gene encoding the same}

The invention cabbage ( Brassica) rapa ssp. The present invention relates to a gene encoding an Argonaute 4 protein isolated from E. pekinensis .

The Agonote 4 (AGO4) gene encodes a protein associated with siRNA-mediated gene silencing as a gene having the PAZ / PIWI domain. The Argonaute family protein is known to be a major factor in the development of post-transcriptional gene silencing (PTGS) in plants. These proteins produce siRNAs of 21 to 22 nucleotides using ribonuclease III enzyme, DICER, and so on, through which PTGS proceeds. In addition, siRNAs, miRNAs, and Piwi-interacting RNAs (piRNAs) are fixed to specific binding pockets and induce silencing to occur as agenote proteins.

The AGO family is highly heterologous and composed of various genes and plays an important role in embryonic development, cell differentiation and transposon silencing (Hock J. and G. Meister. 2008. The Argonaute protein family. Genome Biol . 9: 210). It is divided into AGO subfamily and Piwi subfamily. AGO subfamily expresses variously and binds to siRNA or miRNA to destabilize mRNA or induce PTGS through translational repression. Piwi subfamilies are mostly restricted to the germ line and Piwi proteins are associated with piRNA to promote silencing of mobile genetic elements (Hock J. and G. Meister 2008. The Argonaute protein family. Genome Biol . 9: 210). These are composed of the NH ₂ -terminal PAZ domain and the COOH-terminal PIWI domain which are not precisely characterized. The AGO4 gene is the AGO1 gene (Fagard, M., S. Boutet , JB Morel, C. Bellini, and H. Vaucheret).

The AGO4 gene belonging to the AGO subfamily belongs to Arabidopsis thaliana , is a gene required for DNA methylation induced by 24 nucleotide siRNAs along with RNA polymerases IV and V. AGO4 protein is located close to the nucleus and is associated with the polymerase IV (Pol Ⅳ) subunit NRPD1b, small nuclear RNA binding protein SmD3, trimethylguanosine-capped snRNAs, which are two markers of Cajal body, U2B snRNA binding protein, DNA methyltransferase DRM2 (Li CF, O. Pontes, M. El-Shami, IR Henderson, YV Bernatavichute, SW Chan, T. Lagrange, CS Pikaard, and SE Jacobsen. An Argonaute4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis . Cell . 126: 93-106).

The AGO4 protein plays two major roles in RNA-directed DNA methylation (RdDM). The first function is the catalysis required for siRNA production (Qi, Y., X. He, XJ Wang, O. Kohany, J. Jurka, and GJ Hannon, 2006. Distinct catalytic and non-catalytic roles of Argonaute4 in RNA-directed DNA methylation Nature 443 :..... 1008-1012; Wierzbicki, AT, JR Haag, and CS Pikaard 2008. Noncoding transcription by RNA polymerase PolIVb / PolV mediates transcriptional silencing of overlapping and adjacent genes Cell 135: 635 -648). Second, the AGO4 protein forms an RNA-directed DNA methylation (RdDM) complex with the siRNAs in the target locus and acts as a chromatin remodeling factor. In this complex, the AGO4 protein is known to function as a slicer. When dsRNA is generated by Pol IV and RDR2 (RNA-dependent RNA polymerase 2), siRNA is produced by DCL3 (ribonuclease 3). This 24-nucleotide siRNA binds to the AGO4 protein. Upon chromatin remodeling by DRD1, the AGO4 protein binds to the c-terminal domain (CTD) of Pol V. First, the siRNA of AGO4 binds to DNA after binding to Pol V transcript. (Wierzbicki, AT, JR Haag, and CS Pikaard, 2008). Noncoding transcription by RNA polymerase Pol IVb / Polv mediates transcriptional silencing of overlapping and adjacent genes Cell 135: 635-648.

Thus, when the AGO4 gene involved in the expression of a specific gene is isolated from a Chinese cabbage and induced to induce a change in the internal methylation state, an important change in the inhibition of the transcription activity of the related gene may be caused.

The development of modern molecular biology and the transformation of useful genes are essential for good crop breeding and ongoing adverse weather conditions. However, in these transformations Agrobacterium ( Agrobacterium < RTI ID = 0.0 > Tumefaciens transformed the plant by randomly introducing multiple copies of T-DNA into the target crop, resulting in silencing of multicopy transfectants that are not one copy, resulting in suppression of gene expression do. In addition, the promoter region is methylated by an endogenous defense mechanism to inhibit expression or to be deleted, which is a major obstacle to the commercialization of transgenic crops. Therefore, stable expression of the transgene has been identified as a major factor in botanical and horticultural biotechnology (Hobbs SL, Kpodar P, DeLong CM 1990. The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants Plant Molecular Biology 15, 851-864; Holtorf S, Apel K, Bohlmann H. 1995. Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana . Plant Molecular Biology 29, 637-646). In particular, the silencing of transgenes by multicopy has been reported to stimulate silencing by siRNA chain reaction due to RdDM phenomena in multiple transfected genes (Tang, W., RJ Newton, and DA Weidner. 2007. .. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine J. Exp Bot 58:. 545-554). Accordingly, it is suitable to discover the intrinsic factors (genes) of gene expression control of AGO4 introductory gene which regulates the RdDM pathway of siRNA to induce gene expression. . That is, a competent plant capable of confirming the effective expression of the gene can be cultivated. There is no report on these transgenic plants yet, and it is necessary to keep pace with the future development of plant biotechnology. The AGO4 gene is also an integral part of the RdDM (RNA-directed DNA methylation) complex, which not only functions to activate genes whose expression is suppressed, but also has a close relationship with gene silencing regulation by siRNA. Have. Thus, the regulation of AGO4 genes not only eliminates the inhibition of major genes by biological or abiotic stresses, but also increases the expression stability of useful genes introduced into plant transformation (Qi, Y., X . He, XJ Wang, O. Kohany , J. Jurka, and GJ Hannon 2006. Distinct catalytic and non-catalytic roles of Argonaute4 in RNA-directed DNA methylation Nature 443:... 1008-1012).

In addition, the AGO4 gene has been identified as an essential gene for resistance to one of the plant pathogenic bacteria, Pseudomonas syringae (Agorio, A. and P. Vera. 2007. ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis . Plant Cell 19 (11): 3778-90). Pseudomonas syringae is a pathogenic bacterium that is known as a bacterial black rot fungus such as a black mold of Chinese cabbage, a flower rot, and a tomato bacterium (Inoue, Y. and Y. Takikawa 1999. Grouping Pseudomonas syringae strains by comparison DNA homology at the hrp gene cluster and its neighboring regions. Annals of the Phytopathological Society of Japan . 65 (1): 32-41).

As described above, controlling the AGO4 gene of Chinese cabbage will help to control the black spot disease of Chinese cabbage, but until now, the Chinese cabbage ( Brassica ) and Chinese cabbage rapa ssp. There has been no report on a gene coding for agenote 4 of C. pekinensis .

Accordingly, an object of the present invention is to provide an Argonaute 4 protein derived from Chinese cabbage ( Brassica rapa ssp. Pekinensis ) and a gene encoding the same.

It is another object of the present invention to provide a primer capable of cloning the Agenote 4 gene and a method for isolating the Agenote 4 gene from the cabbage using the primer.

It is another object of the present invention to provide a transgenic Chinese cabbage using the Agenote 4 gene isolated from the Chinese cabbage.

The above object of the present invention can be achieved by a method for preparing a plant, comprising the steps of: preparing a primer capable of cloning the Aegenote 4 gene of Chinese cabbage on the basis of homology with the Aegenomic four base sequence of a previously disclosed vegetable crop; Through the RT-PCR method, the primer thus prepared was used to obtain the fragment and the entire gene of the Agenote 4 gene from the cabbage.

The present invention has the effect of separating and providing the Chinese cabbage-derived Argonaute (Argonaute) 4 gene, and provides for the construction of expression inhibition vector (RNA i vector) using the isolated Argonaute 4 gene and transformed cabbage using the same In addition, by regulating the Aganote 4 gene, it eliminates the inhibition of major genes due to biological or abiotic stress or increases the expression stability of the introduced gene, thereby providing useful breeding material with bacterial black pattern disease resistance of cabbage. Has an excellent effect.

1 is a photograph of a fragment of a gene encoding Agenote 4 amplified from Chinese cabbage by RT-PCR using AGO4-F and AGO4-R, respectively.
FIG. 2 shows a gene encoding Agenote 4 amplified from Chinese cabbage by RT-PCR using AGO4-F and AGO4-R, inserted into pGEM-T easy vector, treated with EcoRI to confirm correct insertion to be.
FIG. 3 shows the sequence of a gene coding for Agenote 4 of Chinese cabbage isolated by RT-PCR using the GenBank BlastN program and Arabidopsis thaliana ) sequence and homology.
Figure 4 shows the sequence of the gene coding for the agenote 4 of Chinese cabbage isolated by RT-PCR using the GenBank BlastX program, Arabidopsis < RTI ID = 0.0 > thaliana ) sequence and homology.
5 shows the nucleotide sequence and amino acid sequence of the gene encoding Agenote 4 of Chinese cabbage separated by RT-PCR.
FIG. 6 shows the results of analysis of a gene encoding Agenote 4 of Chinese cabbage isolated by RT-PCR using the NCBI Conserved Domain program.
Hereinafter, the specific contents of the present invention will be described in detail through preferred embodiments and experimental examples.

The Chinese cabbage ( Brassica rapa ssp. Pekinensis ) used in the present invention was sampled from an aseptic condition in the cabin and used for RNA isolation. Hereinafter, the Agenote 4 gene separation process of the present invention will be described.

Example 1: Agenote 4 gene probe

In order to find a common part of the Agenote 4 gene, the gene of Agenote 4 for each crop has been searched for in the "National Center for Biotechnology Information (NCBI) Genbank". Entrez program was used to collect the entire nucleotide sequence of the gene. As a search method, the genome name was selected as the search term for Argonaute 4 and AGO 4, which is abbreviation of Agenote 4.

The nucleotide sequence of Arabidopsis thaliana , which is a common Chinese cabbage, was selected from among the collected Argonaute 4 genes, and a portion having high homology was screened by BLAST program. F (SEQ ID NO: 1) and AGO4-R (SEQ ID NO: 2) primers are shown in Table 1 below.

primer Base sequence AGO4-F (SEQ ID NO: 1) 5'-ATG GAT GCT ACT ACC GGT AAT G-3 ' AGO4-R (SEQ ID NO: 2) 5'-CTA ACA GAA GAA CAT GGA GTT GG-3 '

Example 2 RNA Separation from Chinese Cabbage

The leaf of Chinese cabbage ( Brassica rapa ssp. Pekinensis ) was disrupted with liquid nitrogen, and 1 ml of trizol (Trizol, Gibco BRL) solution was added, mixed well and allowed to stand at room temperature for 5 minutes. After adding 200 μl of chloroform, the mixture was centrifuged at 4 ° C and 13,000 rpm for 10 minutes, and the supernatant of the mixture was transferred to a new tube. 500 μl of isopropanol was added and centrifuged at 13,000 rpm for 15 minutes to precipitate. Isopropanol was removed, the precipitate was washed with 75% ethanol, dried well without ethanol, and then treated with DEPC (diethyl pyrocarbonate) Dissolved in sterile distilled water.

Example 3: Detection of Agenote 4 gene from Chinese cabbage RNA

In order to isolate the Agenote 4 gene, cDNA was synthesized using oligo dT primer and reverse transcriptase using the obtained RNA as a template, followed by PCR using the primers prepared in [Table 1].

The cDNA synthesis program conditions were as follows: 1 ㎍ of RNA was incubated with oligo dT primer at 70 ° C to remove the RNA secondary structure and then reverse transcribed at 45 ° C for 30 minutes and placed at 94 ° C for 5 minutes to inactivate the used reverse transcriptase. Then, 1 μg of the synthesized cDNA was subjected to PCR using the primer and the tag polymerase described above. The program was subjected to an initial denaturation time of 94 ° C for 2 minutes, followed by 30 seconds at 94 ° C, 30 seconds at 50 ° C, Followed by 40 cycles of 3 min at 72 ° C, final extension at 72 ° C for 10 min, and then the whole reaction was terminated at 4 ° C. In order to effectively isolate a long gene in the above method, a touch-up method in which the annealing step is performed at 0.5 DEG C for each repetition is applied. In the elongation step, (Auto-segment) method is applied.

As a result of the experiment, the fragment of the amplified Agenote 4 gene was confirmed by electrophoresis (FIG. 1). Recombinant plasmid DNA was constructed by cloning the fragments amplified with each primer into a vector pGEM-T easy vector for PCR product to analyze the respective base sequences.

Experimental Example 1: Using recombinant plasmid DNA E. coli  Transformation of DH5α

Escherichia coli DH5α was inoculated into 50 ml of LB and cultured at 37 ° C to OD ₅₇₀ = 0.375-0.400. The culture broth was centrifuged at 3000 rpm for 10 minutes and the resulting precipitate was dissolved in 0.1 M CaCl ₂ solution. I have. After centrifugation at 3000 rpm for 7 minutes, the precipitate was dissolved in 0.1M CaCl ₂ , and the reconstituted plasmid constructed above was mixed with ice for 30 minutes, followed by thermal shock at 42 ° C for 90 seconds. After standing for 10 minutes on ice, 700 μl of LB medium was added and incubated with shaking at 37 ° C for 1 hour. After centrifuging the culture at 12000 rpm for several seconds, the supernatant was removed, leaving only 100 μl. The supernatant was dissolved in the remaining supernatant. Subsequently, a precipitate was dissolved in solid LB medium supplemented with Ampicillin (50 mg / L), X-gal (200 mg / L in NN dimethylformamide) and IPTG (200 mg / L) After the culture, transformants showing resistance to the solid medium and forming white colonies were selected.

Experimental Example 2: Detection of recombinant plasmid DNA and gene insertion

Transformed colonies selected from the above were inoculated in 3 ml of LB medium supplemented with antibiotics, and then cultured at 37 占 폚 for 12 hours. Then, the culture broth was centrifuged at 4 占 폚 and 12,000 rpm for 10 minutes. Solution II (200 μl / ml) was added to the precipitated bacterial mass, followed by vortexing for a few seconds, followed by Solution II (200 μl / ml) and carefully mixed at room temperature And allowed to stand for 5 minutes. After adding Solution III (200 μl / ml), the mixture was mixed well and left on ice for 10 minutes, followed by centrifugation at 12000 rpm at 4 ° C for 10 minutes to obtain supernatant. The obtained supernatant was added with 1/10 of NaOAc and 2.5 times of 100% ethanol, and the plasmid DNA was precipitated at -20 ° C for 2 hours, followed by centrifugation to obtain a precipitate. The precipitate was washed with 70% ethanol and dissolved in sterile distilled water.

To confirm the insertion of the gene coding for Argonaute 4 amplified from Chinese cabbage into the pGEM-T easy vector, restriction enzyme EcoRI was confirmed by treating the isolated recombinant plasmid (Fig. 2). The sequence was examined using T7 and SP6 primers adjacent to the inserted sites.

Experimental Example  3: Agenote  Identification of all four genes

Sequencing of the PCR fragment cloned inside the pGEM-T easy vector for sequencing was performed using the GenBank BLAST program. The nucleotide sequence and amino acid sequence of the 2769 bp product amplified with AGO4-F (SEQ ID NO: 1) and AGO4-R (SEQ ID NO: 2) primers (FIG. 5), Arabidopsis It showed a high homology ahgeo Note 4 gene and 84% of thaliana) (3, 4). That is, RT-PCR using AGO4-F and AOG4-R primers for RNA showed that the entire gene of the agenote 4 gene of Chinese cabbage was amplified (FIG. 5). The PAZ / PIWI domain, which characterizes the Agonote 4 (AGO4) gene, was also identified in the gene isolated using the NCBI Conserved Domain program (FIG. 6). As a result, it was confirmed that the PAZ domain (PAZ_argonaute_like [cd02846], PAZ domain) had an E-value exactly 7.65e-23 and the PIWI domain (PIWI domain, Argonaute-like subfamily) Was 1.09e-155 and the accuracy was high. After determining the start codon and termination codon based on the confirmed nucleotide sequence, it is determined as the entire Aegenote 4 gene isolated from the Chinese cabbage, and its nucleotide sequence is shown in SEQ ID NO: 3 and the amino acid sequence is shown in SEQ ID NO: do.

As described above, the present invention has an effect of providing the Argonaute 4 gene derived from Chinese cabbage, and it has an excellent effect of cultivating a new transgenic Chinese cabbage cultivar using this gene and providing botanical and breeding materials, It is a very useful invention in the breeding industry.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Argonaute 4 of Brassica rapa ssp. pekinensis and a gene encoding          the same <130> 5101 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Brassica rapa ssp. pekinensis <400> 1 atggatgcta ctaccggtaa tg 22 <210> 2 <211> 23 <212> DNA <213> Brassica rapa ssp. pekinensis <400> 2 ctaacagaag aacatggagt tgg 23 <210> 3 <211> 2769 <212> DNA <213> Brassica rapa ssp. pekinensis <400> 3 atggatgcta ctaccggtaa tggagctgag ctcgagtcag caaatgggag tggggtttcc 60 gacgcgctgc ctcctcctcc accggtcata ccgcccaacg tggagcctgt gagggttcaa 120 accgaagttg ctgtgaagaa gaacctaaga gtacctatgg ctcgtccagg ctttggatca 180 aagggtcaaa agattcagct tttgactaat cacttcggag tcaaagttgc aaatcttcag 240 ggattcttct accactacag tgtggcgctc ttctatgatg atgggcgtcc tgttgagcag 300 aaaggggttg gaaggaaagt tcttgacaag gttcatgaga cgtaccactc ggatcttgat 360 ggcaagcagt tcgcctacga tggagagaag acgctgttca cttttggagc tttgcccagc 420 aacaaaatgg atttctccgt ggtccttgag gaagtttctt ccgccaggac taatggaaac 480 gcaagcccca atgggaatga agagccaagt gacggtgata gaaaaagact tcgtcagcct 540 aaccgttcca agagcttcag agtcgagatc agttacgcag caaaaattcc tctccaagct 600 cttgctaacg ctatgcgtgg gcaagaatcc gagaattcac aggaggcaat aagggtgttg 660 gacatcatcc ttcgacagca tgctgctaga caaggttgcc tgcttgtgag acagtccttc 720 ttccacaatg acccgagcaa ctgtgaacct gttggtggta acatcttggg atgtagaggg 780 ttccattcga gtttccggac aacacaaggt ggaatgtcac tcaacatgga tgttacaacc 840 actatgataa tcaagcctgg tccgcttgtt gactttgtca ttgctaacca aggtgcaaag 900 gatcccttta cggttgactg gtctaaggct aaaagaacac ttaagaatct gaggattaaa 960 gtcagcccct caaaccagga gtacaggatc actggcatga gtgacaagcc ttgcagggaa 1020 caaacgtttg agtatagacc aaggaacgca cccaagaatg agaatggaga gtctgaaacc 1080 gttgaaataa cggtgtatga ctacttcctc agagaacgga acctagagtt gcagtactct 1140 gcggatttgc cttgcgtcaa tgttgggagg ccaaagcgac ccacctatat cccccttgag 1200 cactgcacat tgattcccct tcagaggtac acaaaggctc tgaacacttt ccaaagatct 1260 gccctggttg agaaatctag acagaagccg caagagagga tgaatgttct gtcaaaggcg 1320 ctgaaagtga gcaactatga cgctgaacct ctcctgcgtt catgtgggat ttctatcagc 1380 tccaacttta ctcaggtgga gggtcgtgtt ctgccagctc ccaagctgaa aatgggacgt 1440 ggagatgaac tgtttccgag aaatggtcgc tggaatttca acaacaagca atttgtggag 1500 ccaaccaaga ttgataaatg ggctgtagca aatttctctg ctcgctgtaa cgtgcgtcaa 1560 ctggttgatg atctaatccg aattggtgga atgaaaggaa ttgaaattgc tgccccattt 1620 gatgtgtttg aggagggtca tcagtttcgc cgtgctcctc ctatgattcg tgtagagaag 1680 atgtttgaag agatccagtc taagctccct ggtgctcctc agttccttct gtgtctcctc 1740 cccgaaagga agaactgcga catctacggg ccatggaaga agaaaaactt aactgaatac 1800 ggcatcgtta cacaatgcat ggctccggtg aggcagccta atgatcagta tcttaccaac 1860 tgtcttctga agatcaatgc aaagcttggt ggcctcaact ccatgttgag tgtggagcgt 1920 actccagcct ttaccgtcat ttccaaggtt ccaaccatca tccttgggat ggatgtttca 1980 catggatctc ctggacagtc tgatgttccc tccattgctg cggtggtgag ttcaaggcag 2040 tggcctctag tctcgaaata cagagcatct gtacgcacac agccttcaaa ggctgagatg 2100 attgagtcac tcgtcaagaa aaatggaact gaagatgatg gcataatcaa ggagttgctg 2160 gtagatttct acaccagctc gggtaagaga aagccagagc atatcataat cttcagggat 2220 ggtgttagtg aatctcagtt caaccaggtt ctgaacattg agctcgatca gatcattgag 2280 gcctgcaagc ttctagatga gaactggaac ccaaagttcc tcttgttggt ggctcagaag 2340 aatcaccaca ccaagttttt ccagccaaac tctcctgaca acgttccccc agggactatc 2400 atcgacaaca aaatctgcca cccaaagaac aacgacttct atctctgtgc ccacgctggg 2460 atgattggaa caacacgtcc tacgcattac cacgtcctgt atgatgagat tcatttctca 2520 cctgatgagc ttcaagagct cgtccactcg ctctcttacg tgtaccaaag aagcacaacc 2580 gccatctctg ttgttgcacc gatctgctat gcgcacttgg cagctgctca gctaggaacg 2640 ttcatgaagt ttgaagatca gtctgagacg tcctcgagcc atggtggcgt gacagctcca 2700 ggaccagtct ctgttgcgca gctccctaaa ctcaaagaca acgtcgccaa ctccatgttc 2760 ttctgttag 2769 <210> 4 <211> 923 <212> PRT <213> Brassica rapa ssp. pekinensis <400> 4 Met Asp Ala Thr Thr Gly Asn Gly Ala Glu Leu Glu Ser Ala Asn Gly   1 5 10 15 Ser Gly Val Ser Asp Ala Leu Pro Pro Pro Pro Pro Val Ile Pro Pro              20 25 30 Asn Val Glu Pro Val Arg Val Gln Thr Glu Val Ala Val Lys Lys Asn          35 40 45 Leu Arg Val Pro Met Ala Arg Pro Gly Phe Gly Ser Lys Gly Gln Lys      50 55 60 Ile Gln Leu Leu Thr Asn His Phe Gly Val Lys Val Ala Asn Leu Gln  65 70 75 80 Gly Phe Phe Tyr His Tyr Ser Val Ala Leu Phe Tyr Asp Asp Gly Arg                  85 90 95 Pro Val Glu Gln Lys Gly Val Gly Arg Lys Val Leu Asp Lys Val His             100 105 110 Glu Thr Tyr His Ser Asp Leu Asp Gly Lys Gln Phe Ala Tyr Asp Gly         115 120 125 Glu Lys Thr Leu Phe Thr Phe Gly Ala Leu Pro Ser Asn Lys Met Asp     130 135 140 Phe Ser Val Val Leu Glu Glu Val Ser Ser Ala Arg Thr Asn Gly Asn 145 150 155 160 Ala Ser Pro Asn Gly Asn Glu Glu Pro Ser Asp Gly Asp Arg Lys Arg                 165 170 175 Leu Arg Gln Pro Asn Arg Ser Lys Ser Phe Arg Val Glu Ile Ser Tyr             180 185 190 Ala Ala Lys Ile Pro Leu Gln Ala Leu Ala Asn Ala Met Arg Gly Gln         195 200 205 Glu Ser Glu Asn Ser Gln Lys Ala Ile Arg Val Leu Asp Ile Ile Leu     210 215 220 Arg Gln His Ala Ala Arg Gln Gly Cys Leu Leu Val Arg Gln Ser Phe 225 230 235 240 Phe His Asn Asp Pro Ser Asn Cys Glu Pro Val Gly Gly Asn Ile Leu                 245 250 255 Gly Cys Arg Gly Phe His Ser Ser Phe Arg Thr Thr Gln Gly Gly Met             260 265 270 Ser Leu Asn Met Asp Val Thr Thr Thr Met Ile Ile Lys Pro Gly Pro         275 280 285 Leu Val Asp Phe Val Ile Ala Asn Gln Gly Ala Lys Asp Pro Phe Thr     290 295 300 Val Asp Trp Ser Lys Ala Lys Arg Thr Leu Lys Asn Leu Arg Ile Lys 305 310 315 320 Val Ser Pro Ser Asn Gln Glu Tyr Arg Ile Thr Gly Met Ser Asp Lys                 325 330 335 Pro Cys Arg Glu Gln Thr Phe Glu Tyr Arg Pro Arg Asn Ala Pro Lys             340 345 350 Asn Glu Asn Gly Glu Ser Glu Thr Val Glu Ile Thr Val Tyr Asp Tyr         355 360 365 Phe Leu Arg Glu Arg Asn Leu Glu Leu Gln Tyr Ser Ala Asp Leu Pro     370 375 380 Cys Val Asn Val Gly Arg Pro Lys Arg Pro Thr Tyr Ile Pro Leu Glu 385 390 395 400 His Cys Thr Leu Ile Pro Leu Gln Arg Tyr Thr Lys Ala Leu Asn Thr                 405 410 415 Phe Gln Arg Ser Ser Ala Leu Val Glu Lys Ser Arg Gln Lys Pro Gln Glu             420 425 430 Arg Met Asn Val Leu Ser Lys Ala Leu Lys Val Ser Asn Tyr Asp Ala         435 440 445 Glu Pro Leu Leu Arg Ser Ser Cys Gly Ile Ser Ile Ser Ser Asn Phe Thr     450 455 460 Gln Val Glu Gly Arg Val Leu Pro Ala Pro Lys Leu Lys Met Gly Arg 465 470 475 480 Gly Asp Glu Leu Phe Pro Arg Asn Gly Arg Trp Asn Phe Asn Asn Lys                 485 490 495 Gln Phe Val Glu Pro Thr Lys Ile Asp Arg Trp Ala Val Ala Asn Phe             500 505 510 Ser Ala Arg Cys Asn Val Arg Gln Leu Val Asp Asp Leu Ile Arg Ile         515 520 525 Gly Gly Met Lys Gly Ile Glu Ile Ala Ala Pro Phe Asp Val Leu Glu     530 535 540 Glu Gly His Gln Phe Arg Arg Ala Pro Pro Met Ile Arg Val Glu Lys 545 550 555 560 Met Phe Glu Glu Ile Gln Ser Lys Leu Pro Gly Ala Pro Gln Phe Leu                 565 570 575 Leu Cys Leu Leu Pro Glu Arg Lys Asn Cys Asp Ile Tyr Gly Pro Trp             580 585 590 Lys Lys Lys Asn Leu Thr Glu Tyr Gly Ile Val Thr Gln Cys Met Ala         595 600 605 Pro Val Arg Gln Pro Asn Asp Gln Tyr Leu Thr Asn Cys Leu Leu Lys     610 615 620 Ile Asn Ala Lys Leu Gly Gly Leu Asn Ser Met Leu Ser Val Glu Arg 625 630 635 640 Thr Pro Ala Phe Thr Val Ile Ser Lys Val Pro Thr Ile Ile Leu Gly                 645 650 655 Met Gly Val Ser His Gly Ser Pro Gly Gln Ser Asp Val Ser Ser             660 665 670 Ala Ala Val Val Ser Ser Arg Gln Trp Pro Leu Val Ser Lys Tyr Arg         675 680 685 Ala Ser Val Arg Thr Gln Pro Ser Lys Ala Glu Met Ile Glu Ser Leu     690 695 700 Val Lys Lys Asn Gly Thr Glu Asp Asp Gly Ile Ile Lys Glu Leu Leu 705 710 715 720 Val Asp Phe Tyr Thr Ser Ser Gly Lys Arg Lys Pro Glu His Ile Ile                 725 730 735 Ile Phe Arg Asp Gly Val Ser Glu Ser Gln Phe Asn Gln Val Leu Asn             740 745 750 Ile Glu Leu Asp Glu Ile Ile Glu Ala Cys Lys Leu Leu Asp Glu Asn         755 760 765 Trp Asn Pro Lys Phe Leu Leu Leu Val Ala Gln Lys Asn His His Thr     770 775 780 Lys Phe Phe Gln Pro Asn Ser Pro Asp Asn Val Pro Pro Gly Thr Ile 785 790 795 800 Ile Asp Asn Lys Ile Cys His Pro Lys Asn Asn Asp Phe Tyr Leu Cys                 805 810 815 Ala His Ala Gly Met Ile Gly Thr Thr Arg Pro Thr His Tyr His Val             820 825 830 Leu Tyr Asp Glu Ile His Phe Ser Pro Asp Glu Leu Gln Glu Leu Val         835 840 845 His Ser Leu Ser Tyr Val Tyr Gln Arg Ser Thr Thr Ala Ile Ser Val     850 855 860 Val Ala Pro Ile Cys Tyr Ala His Leu Ala Ala Ala Gln Leu Gly Thr 865 870 875 880 Phe Met Lys Phe Glu Asp Gln Ser Glu Thr Ser Ser Ser His Gly Gly                 885 890 895 Val Thr Ala Pro Gly Pro Val Val Ser Ala Gln Leu Pro Lys Leu Lys             900 905 910 Asp Asn Val Ala Asn Ser Met Phe Phe Cys ***         915 920

Claims (5)

SEQ ID NO: 1 and consisting of Chinese cabbage of the nucleotide sequence of SEQ ID NO: 2 (Brassica rapa ssp. Pekinensis) ahgeo notes (Argonaute) 4 primers derived from the gene encoding the protein.
A gene coding for the Argonaute 4 protein derived from cabbage comprising the nucleotide sequence of SEQ ID NO: 3.
Argonaute 4 protein from cabbage comprising the amino acid sequence of SEQ ID NO: 4.
A recombinant plasmid prepared by cloning the Argonaute 4 gene of claim 2 into a pGEM-T easy vector.
Transgenic cabbage expressed by introduction of Argonaute 4 gene derived from Chinese cabbage.

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