KR20100130425A - Histone deacetylase protein of brassica rapa ssp. pekinensis and a gene encoding the same - Google Patents

Abstract

PURPOSE: A Brassica rapa ssp. Pekinensis-derived histone deacetylase protein is provided to suppress histone methylation and plant tumor generation. CONSTITUTION: A Brassica rapa ssp. Pekinensis-derived histone deaceylase protein has an amino acid sequence of sequence number 6. A gene encoding the protein has a nucleic acid sequence of sequence number 5. A recombinant vector, pB7GWIWG2(II)-HDI, contains histone diacetylase gene. A transformed Brassica rapa ssp. Pekinensis is prepared using a transformant. A method for isolating the gene comprises: a step of preparing a primer of sequence numbers 1-2; a step of performing RT-PCR to isolate a gene from a histone deacetylase protein; a step of confirming the total sequence of histoen deacetaylase using the isolated gene.

Description

Histone deacetylase protein derived from Chinese cabbage and genes encoding the same {HISTONE DEACETYLASE PROTEIN OF BRASSICA RAPA SSP. PEKINENSIS AND A GENE ENCODING THE SAME}

The present invention is a histone which is involved in the methylation process of DNA and histones of Chinese cabbage (Brassica rapa ssp. Pekinensis) diacetyl la azepin method for separating proteins from cabbage (histone deacetylase) and, separated histone diacetyl la kinase and encoding the It's about genes.

The study of DNA methylation in order to elucidate the mechanism of electron regulation is called epigenetics. Scientists are doing their best to use information from research to apply it to medicine or to develop new drugs. To do this, however, the functions and regulatory mechanisms of the genes that make proteins must first be identified. Until now, it is known that changes in traits are caused by changes in DNA sequence and recombination. However, even if the DNA sequence does not change, the function of the gene changes, and it is found that this change is transmitted from the parent to the offspring. Epigenetics is a new field of study that discovers how this phenomenon, namely changes in function such as gene expression, occurs without changing the DNA base sequence.

An important phenomenon from a general genetic point of view is the mutation of a base change, but in epigenetics a methylation process that attaches a methyl group to a base. The presence of two bases, C and G, side by side in the nucleotide sequence of the genome is called CpG. Cytosine tends to be methylated in this arrangement, so in the human genome, 3-4% of all cytosine is methylated. Methylation of genes suppresses the transcriptional process by histone modification or chromatin remodeling rather than methyl transferase activity.

Epigenetic regulatory mechanisms for the transcription of genes are closely linked to different mechanisms: methylation of DNA, methylation and deacetylation of histones, and chromatin remodeling. Among the three, recent reports suggest that methylation of histones plays an important role in cell division and gene expression. One group of genes that are expressed early in the embryonic development stage is the HOX group, which forms patterns in the body and then potentially loses function forever. Another gene family that plays an important role in the potential of the HOX gene group is the polycomb gene family. Mutations in this family of genes result in abnormal development of the embryo because HOX is not latent.

Eukaryotic DNA is wound twice in a basic protein called histones and stored in a narrow nucleus space. In order to utilize such compressed DNA as genetic information, cells have been expected to have a mechanism for modifying and controlling chromatin structure. Recently, the nucleosome structure is modified to access transcription factors and DNA. Increasing chromatin remodeling factors (SWI / SNF, RSC, NURF, NRD, etc.) and histone acetyltransferases (HAs) and histone deacetylases that regulate the acetylation state of histones (histone deacetylases; HDs) have been found to be important regulators. In particular, the process of neutralizing the positive charge of the lysine residue at the amino terminus of the histone by acetylation or inducing the charge again by deacetylation is a reversible histone modification reaction, inducing the equilibrium of the histone acetylation level, thereby turning on / off the gene expression switch. Adjust the off.

Histone deacetylase is an important factor responsible for chromatin restructuring reactions. To date, worldwide research in this field has been the mainstream of cloning of new histone deacetyllanges and the isolation and function interpretation of related factors. In the future, however, the analysis of the network of chromatin structural factor complexes through specific interactions with chromatin structural factors (HAT, nucloesome remodeling factor, histone chaperon, etc.) related to histone deacetylases will be performed at various levels. Chromatin restructuring transcript studies will be developed. This interpretation will answer questions about how histone deacetylases are recruited to specific DNA to regulate the expression of specific genes.

On the other hand, the regulation of histone deacetylases associated with diseases (cancer, leukemia, angiogenesis, etc.) will be of increasing importance. In particular, if the high acetylation of chromatin inhibits cell proliferation and promotes differentiation, histone deacetylases play a critical role in inducing proliferation through deacetylation of histones (Kim MS, Kwon HJ, Lee). YM, Baek JH, Jang JE, Lee SW, Moon EJ, Kim HS, Chung HY, Kim CW, Kim KW, 2001, Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes.Nature Medicine, in press . This fact is supported by the results of inhibition of cell proliferation and angiogenesis when treated with histone deacetylase inhibitors.

Therefore, histone acetylases are not only important research subjects in the interpretation of functional roles as gene expression inhibitors, but also important as target molecules for the development of new anticancer drugs. Among the histone deacetylase inhibitors, FR901288 and MS-275 have shown wide anticancer activity in animal studies, and clinical trials are underway in NCI (Kwon HJ, 2001, Histone deacetylase, a key covalent modifier of histone and regulator of gene expression). Biochemistry News Vol. 21, No. 1).

However, there is no report on the gene encoding histone deacetylase in Chinese cabbage.

The present inventors have identified genes encoding histone polyacetylases derived from Chinese cabbage, using them to identify the role of histone deacetylases in Chinese cabbage, and based on this, the functional cabbage with high inhibitory effect on plant tumor development is cultivated. It continued to research to improve public health.

The present invention is to provide a histone deacetylase protein derived from cabbage and genes encoding the same.

The present invention also provides a primer for detecting histone deacetylase protein and a method for separating a gene encoding histone deacetylase from cabbage using the same.

The present invention relates to a histone deacetylase protein derived from cabbage, having the amino acid sequence of SEQ ID NO: 6.

Primer pairs were prepared and used for “histone deacetylase protein” isolation of the invention. RT-PCR was performed to separate the gene corresponding to the histone deacetylase protein from the cabbage using the prepared primer pairs to secure the entire sequence of the gene encoding the histone deacetylase protein.

The histone deacetylase protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 6. Also included within the scope of the invention are variants in which some amino acid sequences at one or more positions are deleted, inserted, nonconservative or conservative substitutions, or a combination thereof.

The present invention also relates to a gene comprising a nucleic acid sequence encoding a histone deacetylase protein derived from cabbage having the amino acid sequence of SEQ ID NO: 6.

The gene encoding the histone deacetylase protein having the amino acid sequence of SEQ ID NO: 6 of the present invention is preferably a gene having a nucleic acid sequence of SEQ ID NO: 5.

Nucleic acid sequences encoding histone deacetylase proteins may be mutated by one or more bases substituted, deleted, inserted, or a combination thereof. Such nucleic acid sequence variants are also within the scope of the present invention.

The invention also relates to a recombinant vector comprising a gene comprising a nucleic acid sequence encoding a histone deacetylase protein.

As used herein, a "recombinant vector" is a genetic construct comprising essential regulatory elements operably linked to express a gene insert, including all conventional vectors, including plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. .

In the vector of the present invention, the nucleic acid sequence encoding histone deacetylase protein is functionally linked to the expression control sequence. For example, a vector may include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and may be prepared in various ways depending on the purpose. have. The vector may comprise a selectable marker. In addition, the vector can self-replicate or integrate into the host DNA. Vectors of the invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.

The invention also relates to a transformant transformed with a recombinant vector comprising a gene comprising a nucleic acid sequence encoding a histone deacetylase protein. Specifically, according to the present invention, using the histone deacetylase protein and the sequence of the gene for it, the study on DNA and histone methylation phenomena can be carried out, and the transformed cabbage with high inhibitory effect on plant tumor development can be made.

Vector introduction into a host cell can be carried out by selecting a method suitable for the host cell. For example, electroporation, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agrobacterial mediated transformation, particle counting, protoplast cytometry, text box It can be carried out by known methods such as sulfate, lipofectamine and the like.

As the host cell, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis) or Staphylococcus (Staphylococcus) Prokaryotic host cells such as, but are not limited to these. In addition, fungi (e.g. Aspergillus ), yeast (e.g. Pichia pastoris , Saccharomyces cerevisiae ), Schizocaromaces ( Cells of eukaryotes such as Schizosaccharomyces , Neurospora crassa , insect cells, plant cells, mammals, etc., but are not limited thereto.

In addition, the gene encoding the histone deacetylase protein of the present invention can be transformed into a plant individual. The transformation into plants is preferably the Agrobacterium method, the plasma cell method, the particle total method and the like. The plant to be transformed is preferably cabbage.

In addition, the present invention is to increase or decrease the level of intracellular expression of histone deacetylase protein having the amino acid sequence of SEQ ID NO: 6 to study the DNA and histone methylation phenomena of plants, preferably cabbage, plant tumor development It provides a functional cabbage with a high inhibitory effect.

For example, intracellular expression levels of histone deacetylase protein can be increased by transforming plants, more specifically cabbage, with recombinant vectors encoding histone deacetylase protein. Recombinant vectors encoding histone deacetylase proteins are as described above. In addition, one or more exogenous nucleic acid sequences may be introduced into the exon of the genomic gene encoding histone deacetylase protein to inactivate the gene to inhibit and block the level of intracellular expression of the histone deacetylase protein. As used herein, the term “exogenous nucleic acid sequence” refers to a nucleic acid sequence that is not normally found in a host cell, and inactivation of a gene may be induced by inserting an exogenous nucleic acid sequence into an exon of a gene genome.

By the method exemplified above, the expression of histone deacetylase protein in plants, preferably cabbage, can be effectively controlled to suit the purpose.

In addition, the present invention comprises the steps of (i) preparing a primer set forth in SEQ ID NO: 1 to 2; (ii) isolating the gene for the histone deacetylase protein by PCR using cDNA synthesized from the RNA of Chinese cabbage, using a primer pair consisting of SEQ ID NOS: 1 to 2; And (iii) identifying the entire sequence of the histone deacetylase gene using the isolated gene.

The steps are as follows.

Prior to the step (i), the sequence for the gene of the gene protein encoding a known histone deacetylase protein for the plant is collected and the gene coding sequence is aligned with the region of high homology among the aligned sequences. The step of selecting may be performed first. At this time, the sequence for the gene of the known histone deacetylase protein for the plant can be obtained from NCBI GenBank and the like, homology comparison can be performed with the naked eye or using a comparison program.

Then, the primer of step (i) is prepared, and a primer pair specific to the region of high homology is prepared. In the present invention, the length of the primer serving as a starting point for copying the template strand is not particularly limited and has 10 to 50, more preferably 10 to 40 base sequences. Specifically, the sequences of the primers used in the present invention are set forth in SEQ ID NOs: 1-2.

In step (ii), RNA isolation and PCR from the sample can be carried out in a general manner. In step (iii), a method of performing RT-PCR using an isolated gene fragment may be used, and the RT-PCR may be performed by a known general method.

The kind of plant which can be used for the histone deacetylase protein isolation by the method of the present invention is not particularly limited. For example, various plants belonging to food crops, vegetable crops, special crops, fruit trees, flowers and feed crops can be used as samples.

Specifically, the cabbage ( Brassica rapa ssp. Pekinensis ) used in the separation of histone deacetylase protein in the present invention was isolated from RNA by taking a sample in the sterile state in the cabin.

The present invention also provides a primer for detecting histone deacetylase protein selected from SEQ ID NO: 1 to 2.

Since the primers provided in the present invention selectively recognize regions of high homology between histone deacetylase proteins of plants, they are primers that can be efficiently used for detection of histone deacetylase proteins in various plants.

The forward and reverse primer pairs of SEQ ID NOS: 1 to 2 initiate cDNA synthesis in the presence of an enzyme and four different nucleoside triphosphates for polymerization in the appropriate buffer and temperature.

Primer sequences of the present invention may comprise a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Labels include enzymes such as horseradish peroxidase, alkaline phosphatase and the like; Radioisotopes such as ³² P; fluorescent molecules such as cy3 and cy5; Chemical groups such as biotin are exemplified.

The method of the present invention can effectively separate histone deacetylase protein genes from plants. In addition, since the histone deacetylase protein according to the present invention is related to DNA and histone methylation and the inhibitory effect of plant tumor development, plant tumors are generated when plant transformation is made by using the down-regulation vector. Functional cabbage with a high inhibitory effect can be provided. It could also further benefit the provision of cabbage varieties useful for public health.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Preparation of Primer for Isolating Histone Deacetylase Protein Gene

In order to find a common part of histone deacetylase protein gene, previously published histologic deacetylase protein gene was searched in the National Center for Biotechnology Information (NCBI) Genbank. Entrez program was used to collect the entire sequence of the gene. As a search method, histone deacetylase was selected as a keyword.

Among the genes of histone deacetylase protein collected, the nucleotide sequence of Arabidopsis thaliana of the cabbage family, which is a model plant, was selected, and the part showing much homology of nucleotide sequence was found by the BLAST program, and then the primer was mainly focused on the part. Was produced. The sequences of the prepared primers are as described in Table 1.

primer Sequence HD-F (SEQ ID NO: 1) 5'-ATG GAC ACC GGA GGA AAC TCG C-3 ' HD-R (SEQ ID NO: 2) 5'-TTA GGT TTT AGG AAA CAC TTG ATC-3 '

Example 2: Gene Isolation of Histone Deacetylase Proteins

The isolation of RNA from Chinese cabbage ( Brassica rapa ssp. Pekinensis ) was performed using Trizol reagent provided by Gibco BRL. The leaves of Chinese cabbage were crushed with liquid nitrogen, 1 ml Trizol solution was added, mixed well, and left at room temperature for 5 minutes. Next, 200 μl of chloroform was added and mixed, followed by further centrifugation at 13,000 rpm for 10 minutes at 4 ° C., and the supernatant of the mixture was transferred to a new tube. 500 μl of isopropanol was added and precipitated by centrifugation at 13,000 rpm for 15 minutes. Next, the isoprophenol was removed and the precipitate was washed with 75% ethanol, dried well without ethanol, and then dissolved in DEPC-treated sterile distilled water.

Isolation of histone deacetylase protein gene was obtained by using RNA as a template and cDNA synthesized using dT primer and reverse transcriptase. The cDNA synthesis program conditions were 1 μg of RNA was raised with the dT primer at 70 ° C. to remove the RNA secondary structure, reverse transcribed at 45 ° C. for 30 minutes, and placed at 94 ° C. for 5 minutes to inactivate the used reverse transcriptase. After that, PCR was performed using the prepared primer and tag polymerase using 1 microgram of cDNA. The program allowed initial denaturation time at 94 ° C. for 2 minutes, 30 seconds at 94 ° C., 30 seconds at 56 ° C. 40 cycles were performed at 72 ° C. for 2 minutes and finally extended at 72 ° C. for 10 minutes to maintain 4 ° C. to complete the entire reaction.

The fragments amplified with the respective primers were cloned into the vector pGem-T easy vector for PCR products to analyze their respective nucleotide sequences, and the restriction enzyme recognition sequence Eco RI was used to confirm that the correct gene was inserted. The result is shown in FIG. Subsequently, sequences were analyzed using T7 and SP6 primers that bind to the T7 promoter and the SP6 promoter close to the site where the gene was inserted.

Example 3: Confirming the entire sequence of the histone deacetylase protein gene

Sequencing of the PCR fragment cloned into the pGem-T easy vector for sequencing was performed using the GenBank BLAST program. As a result, the nucleotide sequence (figure 1) and amino acid sequence of 1509 bp amplified by HD-F (SEQ ID NO: 1) and HD-R (SEQ ID NO: 2) primers were analyzed. Arabidopsis thaliana and rice ( Oryza) sativa ) showed homology with the histone deacetylase gene of plants (see FIGS. 3 and 4), especially 86% homology with the histone deacetylase 1 gene of Arabidopsis. In other words, RT-PCR was performed on the RNA using HD-F (SEQ ID NO: 1) and HD-R (SEQ ID NO: 2) primers to confirm that the entire gene of histone deacetylase gene in Chinese cabbage was amplified. there was.

In addition, using the NCBI Conserved Domain program (see FIG. 5), the histone deacetylase gene was isolated and the histone deacetylase domain and yeast histone deacetylase and acetoin using protein were included. Deacetylases, including yeast histone deacetylase and acetoin utilization protein were confirmed to have high accuracy by accurately including their respective E-values as 7e-85 and 1e-85. Based on the identified histone deacetylase gene sequence, the start codon and the end codon were determined, and then, the total codon was determined from the total histone deacetylase gene isolated from the cabbage, and the amino acid sequence of the histone deacetylase protein was determined. Is shown in SEQ ID NO: 6. And the gene sequence for the protein is shown in SEQ ID NO: 5.

Example 4: Recombinant vector construction using histone deacetylase protein gene

To make the plant expression vector in gene expression for the vector, the expression vector was used to inhibit RNA i pB7GWIWG2 (II) vector (VIB-Ghent University, USA), which contains a ^{(invitrogen, USA) Gateway ® system} . For expression inhibition RNA i vector, for histological inhibition, the histone deacetylase domain and the histone deacetylase domain were isolated from the histone deacetylase gene based on the analysis results using the NCBI Conserved Domain program (see FIG. 5). A primer was prepared to effectively inhibit the deacetylases (including yeast histone deacetylase and acetoin utilization protein) of yeast histone deacetylase and acetoin using protein. The sequences of the prepared primers are as described in Table 2.

primer Sequence HD-iF
(SEQ ID NO: 3) 5'-AGC GTG TTC TGT ACG TTG ATA TC-3 ' HD-iR
(SEQ ID NO: 4) 5'-GCA ACT CCA GTC TCG TAG CAC C-3 '

In Example 2, the nucleotide sequence of the histone deacetylase protein gene cloned in the pGem-T easy vector for sequencing was isolated by PCR with a primer prepared. Synthesis program conditions were PCR using primers and tag polymerase prepared using 1 microgram of plasmid. The program was initially initiated at 94 ° C. for 2 minutes for the expression inhibitory RNA i vector fragment using the primers shown in Table 2. After the denaturation time, 40 cycles were performed at 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute. It was.

As a result, 453 bp was obtained for the expression inhibitory RNA i vector fragment as shown in FIG. 6. The fragments amplified with the respective primers were then cloned into each of the pCR 8 / GW / TOPO TA vectors to apply the Gateway ^® system (invitrogen, USA) and restriction enzyme recognition to confirm that the correct gene was inserted. The sequence Eco RI was used, as shown in FIG. 7. Subsequently, sequences were analyzed using T7 and T3 primers that bind to the T7 promoter and the T3 promoter close to the site where the gene was inserted. After Gateway ^® LR Clonase ^™ II enzyme mix using (invitrogen, USA), the expression suppressing RNA i vector was to be inserted into the pB7GWIWG2 (II) vector with sense and antisense direction. This recombinant vector was named pB7GWIWG2 (II) -HDI and is shown in FIG. 8. In the vector of FIG. 8, Sm / SpR is streptomycin / spectinomycin resistant Sm / SpR gene, LB and RB represent left and right borders of T-DNA, BAR is herbicide resistant BAR gene, and p35S is cauliflower Mosaic virus (CaMV) 35S promoter representing p35S, T35S is the T35S representing the terminator of CaMV 35S. CmR is a chloramphenicol resistant CmR gene.

1 is a photograph of electrophoresis of fragments of genes encoding histone deacetylase amplified from cabbage by RT-PCR using HD-F and HD-R.

Figure 2 shows a gene encoding the histone deacetylase amplified from cabbage by RT-PCR using HD-F (SEQ ID NO: 1) and HD-R (SEQ ID NO: 2) pGem-T easy vector This is a picture of Eco RI processed to confirm correct insertion.

3 is a result of homology comparison between Arabidopsis thaliana sequences using the GenBank BlastX program for sequences of genes encoding histone deacetylase of Chinese cabbage isolated by RT-PCR.

FIG. 4 shows the results of homology comparison between Oryza sativa sequences using GenBank BlastX program for a sequence of genes encoding histone deacetylase of Chinese cabbage separated by RT-PCR.

5 is a result of analyzing the gene encoding histone deacetylase of Chinese cabbage separated by RT-PCR using the NCBI Conserved Domain program.

Figure 6 is a photograph of the electrophoresis of RNA i vector fragments of genes encoding histone deacetylase amplified from Chinese cabbage by PCR using each HD-iF and HD-iR.

Figure 7 shows the gene encoding histone deacetylase of Chinese cabbage by PCR using HD-iF (SEQ ID NO: 3) and HD-iR (SEQ ID NO: 4) to pCR 8 / GW / TOPO TA vector After inserting the product, Eco RI was processed to confirm the correct insertion.

8 is a schematic diagram showing pB7GWIWG2 (II) -HDI, an expression inhibitory RNA i vector that regulates a gene encoding histone deacetylase.

<110> UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY <120> HISTONE DEACETYLASE PROTEIN OF Brassica rapa ssp. pekinensis AND          A GENE ENCODING THE SAME <130> P09-139 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HD-F Primer <400> 1 atggacaccg gaggaaactc gc 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HD-R Primer <400> 2 ttaggtttta ggaaacactt gatc 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HD-i-F Primer <400> 3 agcgtgttct gtacgttgat atc 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HD-i-R Primer <400> 4 gcaactccag tctcgtagca cc 22 <210> 5 <211> 1509 <212> DNA <213> Brassica rapa ssp. pekinensis <220> <221> CDS (1) .. (1506) <400> 5 atg gac acc gga gga aac tcg ctg gcc tct gga cca gac ggt gtg aag 48 Met Asp Thr Gly Gly Asn Ser Leu Ala Ser Gly Pro Asp Gly Val Lys   1 5 10 15 agg aaa gtg tgc tac ttc tac gac cca gag gtt ggg aac tac tac tac 96 Arg Lys Val Cys Tyr Phe Tyr Asp Pro Glu Val Gly Asn Tyr Tyr Tyr              20 25 30 ggc caa ggc cac ccc atg aag cct cac cgc atc cgc atg acg cac gct 144 Gly Gln Gly His Pro Met Lys Pro His Arg Ile Arg Met Thr His Ala          35 40 45 ctc ctc gct cac tac ggt ctc ctc cag cac atg caa gtc ctc aag cct 192 Leu Leu Ala His Tyr Gly Leu Leu Gln His Met Gln Val Leu Lys Pro      50 55 60 ttc ccc gcc cgt gac cgt gac ctc tgc cgc ttc cac gcc gac gac tac 240 Phe Pro Ala Arg Asp Arg Asp Leu Cys Arg Phe His Ala Asp Asp Tyr  65 70 75 80 gtc tcc ttt ctc cgc agc att acg ccc gag act cag cag gat cag atc 288 Val Ser Phe Leu Arg Ser Ile Thr Pro Glu Thr Gln Gln Asp Gln Ile                  85 90 95 cgc cag ctc aag cgg ttc aac gtt ggt gaa gac tgt cct gtc ttt gac 336 Arg Gln Leu Lys Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe Asp             100 105 110 ggg ctt tat tcc ttt tgt cag acg tat gct ggt ggc tct gtt ggt ggt 384 Gly Leu Tyr Ser Phe Cys Gln Thr Tyr Ala Gly Gly Ser Val Gly Gly         115 120 125 tct gtt aag ctt aac cat ggg ctt tgt gat att gct att aac tgg gct 432 Ser Val Lys Leu Asn His Gly Leu Cys Asp Ile Ala Ile Asn Trp Ala     130 135 140 ggt ggt ctt cat cat gct aag aag tgt gag gcc tct ggc ttc tgc tac 480 Gly Gly Leu His His Ala Lys Lys Cys Glu Ala Ser Gly Phe Cys Tyr 145 150 155 160 gtt aat gat atc gtc tta gct atc ctt gag ctg ctt aag cag cat gag 528 Val Asn Asp Ile Val Leu Ala Ile Leu Glu Leu Leu Lys Gln His Glu                 165 170 175 cgt gtt ctg tac gtt gat atc gac att cac cac ggg gac gga gtg gag 576 Arg Val Leu Tyr Val Asp Ile Asp Ile His His Gly Asp Gly Val Glu             180 185 190 gag gcg ttt tac gct acc gac agg gtc atg acc gtc tcg ttc cat aag 624 Glu Ala Phe Tyr Ala Thr Asp Arg Val Met Thr Val Ser Phe His Lys         195 200 205 ttc gga gac tac ttc ccc ggt aca ggc cac ata caa gac att ggc tac 672 Phe Gly Asp Tyr Phe Pro Gly Thr Gly His Ile Gln Asp Ile Gly Tyr     210 215 220 gga agt gga aag tac tat tca ctc aac gta cca ctg gac gat ggt atc 720 Gly Ser Gly Lys Tyr Tyr Ser Leu Asn Val Pro Leu Asp Asp Gly Ile 225 230 235 240 gac gat gag agc tat cat ctc ttg ttc aaa ccc atc atg ggg aaa gtg 768 Asp Asp Glu Ser Tyr His Leu Leu Phe Lys Pro Ile Met Gly Lys Val                 245 250 255 atg gaa att ttc aaa cca ggt gcg gtc gta ctc caa tgc ggc gct gat 816 Met Glu Ile Phe Lys Pro Gly Ala Val Val Leu Gln Cys Gly Ala Asp             260 265 270 tcc ttg tct ggc gat agg ctg gga tgc ttt aac ctc tcc atc aaa ggc 864 Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu Ser Ile Lys Gly         275 280 285 cat gcc gag tgt gtc aag ttc atg aga tct ttc aac gtt ccg tta ctg 912 His Ala Glu Cys Val Lys Phe Met Arg Ser Phe Asn Val Pro Leu Leu     290 295 300 ctc ctt ggt ggt ggt ggt tac acc atc cgc aac gtt gcg cgt tgc tgg 960 Leu Leu Gly Gly Gly Gly Tyr Thr Ile Arg Asn Val Ala Arg Cys Trp 305 310 315 320 tgc tac gag act gga gtt gcg ctt ggg atc gag gtc gag gac aag atg 1008 Cys Tyr Glu Thr Gly Val Ala Leu Gly Ile Glu Val Glu Asp Lys Met                 325 330 335 ccg gag cat gag tac tat gag tac ttt ggt ccg gac tat acg ctc cac 1056 Pro Glu His Glu Tyr Tyr Glu Tyr Phe Gly Pro Asp Tyr Thr Leu His             340 345 350 gtt gct ccg agt aac atg gag aac aag aac tct cgg cag atg ctc gaa 1104 Val Ala Pro Ser Asn Met Glu Asn Lys Asn Ser Arg Gln Met Leu Glu         355 360 365 gtg att cgt aat gac ttg ctc cag aat ctc tcg aag ctt cag cat gct 1152 Val Ile Arg Asn Asp Leu Leu Gln Asn Leu Ser Lys Leu Gln His Ala     370 375 380 cct agt gtg ccg ttt cag gaa agg ccg cca gat act gag act cct gag 1200 Pro Ser Val Pro Phe Gln Glu Arg Pro Pro Asp Thr Glu Thr Pro Glu 385 390 395 400 gta gat gaa gac caa gaa gat gga gat aaa aga tgg gat gca gat tct 1248 Val Asp Glu Asp Gln Glu Asp Gly Asp Lys Arg Trp Asp Ala Asp Ser                 405 410 415 gac atg gat gta gat gat gat cgt aaa cct ata cca agc aga gtg aaa 1296 Asp Met Asp Val Asp Asp Asp Arg Lys Pro Ile Pro Ser Arg Val Lys             420 425 430 aga gaa gct gtt gag cct gat gga aag gac aag gat gga gtg aaa gga 1344 Arg Glu Ala Val Glu Pro Asp Gly Lys Asp Lys Asp Gly Val Lys Gly         435 440 445 gtg atg gag cgt ggg aaa ggt ttt gat gtg ggc atg gag gag agt gga 1392 Val Met Glu Arg Gly Lys Gly Phe Asp Val Gly Met Glu Glu Ser Gly     450 455 460 agc acc acc aag gtt tca gga gta aac tca gtg gga atg gat gaa gca 1440 Ser Thr Thr Lys Val Ser Gly Val Asn Ser Val Gly Met Asp Glu Ala 465 470 475 480 ggt gtg aaa atg gaa gag gaa ggc aca aca aac aag tca ggg gga gat 1488 Gly Val Lys Met Glu Glu Glu Gly Thr Thr Asn Lys Ser Gly Gly Asp                 485 490 495 caa gtg ttt cct aaa acc taa 1509 Gln Val Phe Pro Lys Thr             500 <210> 6 <211> 502 <212> PRT <213> Brassica rapa ssp. pekinensis <400> 6 Met Asp Thr Gly Gly Asn Ser Leu Ala Ser Gly Pro Asp Gly Val Lys   1 5 10 15 Arg Lys Val Cys Tyr Phe Tyr Asp Pro Glu Val Gly Asn Tyr Tyr Tyr              20 25 30 Gly Gln Gly His Pro Met Lys Pro His Arg Ile Arg Met Thr His Ala          35 40 45 Leu Leu Ala His Tyr Gly Leu Leu Gln His Met Gln Val Leu Lys Pro      50 55 60 Phe Pro Ala Arg Asp Arg Asp Leu Cys Arg Phe His Ala Asp Asp Tyr  65 70 75 80 Val Ser Phe Leu Arg Ser Ile Thr Pro Glu Thr Gln Gln Asp Gln Ile                  85 90 95 Arg Gln Leu Lys Arg Phe Asn Val Gly Glu Asp Cys Pro Val Phe Asp             100 105 110 Gly Leu Tyr Ser Phe Cys Gln Thr Tyr Ala Gly Gly Ser Val Gly Gly         115 120 125 Ser Val Lys Leu Asn His Gly Leu Cys Asp Ile Ala Ile Asn Trp Ala     130 135 140 Gly Gly Leu His His Ala Lys Lys Cys Glu Ala Ser Gly Phe Cys Tyr 145 150 155 160 Val Asn Asp Ile Val Leu Ala Ile Leu Glu Leu Leu Lys Gln His Glu                 165 170 175 Arg Val Leu Tyr Val Asp Ile Asp Ile His His Gly Asp Gly Val Glu             180 185 190 Glu Ala Phe Tyr Ala Thr Asp Arg Val Met Thr Val Ser Phe His Lys         195 200 205 Phe Gly Asp Tyr Phe Pro Gly Thr Gly His Ile Gln Asp Ile Gly Tyr     210 215 220 Gly Ser Gly Lys Tyr Tyr Ser Leu Asn Val Pro Leu Asp Asp Gly Ile 225 230 235 240 Asp Asp Glu Ser Tyr His Leu Leu Phe Lys Pro Ile Met Gly Lys Val                 245 250 255 Met Glu Ile Phe Lys Pro Gly Ala Val Val Leu Gln Cys Gly Ala Asp             260 265 270 Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu Ser Ile Lys Gly         275 280 285 His Ala Glu Cys Val Lys Phe Met Arg Ser Phe Asn Val Pro Leu Leu     290 295 300 Leu Leu Gly Gly Gly Gly Tyr Thr Ile Arg Asn Val Ala Arg Cys Trp 305 310 315 320 Cys Tyr Glu Thr Gly Val Ala Leu Gly Ile Glu Val Glu Asp Lys Met                 325 330 335 Pro Glu His Glu Tyr Tyr Glu Tyr Phe Gly Pro Asp Tyr Thr Leu His             340 345 350 Val Ala Pro Ser Asn Met Glu Asn Lys Asn Ser Arg Gln Met Leu Glu         355 360 365 Val Ile Arg Asn Asp Leu Leu Gln Asn Leu Ser Lys Leu Gln His Ala     370 375 380 Pro Ser Val Pro Phe Gln Glu Arg Pro Pro Asp Thr Glu Thr Pro Glu 385 390 395 400 Val Asp Glu Asp Gln Glu Asp Gly Asp Lys Arg Trp Asp Ala Asp Ser                 405 410 415 Asp Met Asp Val Asp Asp Asp Arg Lys Pro Ile Pro Ser Arg Val Lys             420 425 430 Arg Glu Ala Val Glu Pro Asp Gly Lys Asp Lys Asp Gly Val Lys Gly         435 440 445 Val Met Glu Arg Gly Lys Gly Phe Asp Val Gly Met Glu Glu Ser Gly     450 455 460 Ser Thr Thr Lys Val Ser Gly Val Asn Ser Val Gly Met Asp Glu Ala 465 470 475 480 Gly Val Lys Met Glu Glu Glu Gly Thr Thr Asn Lys Ser Gly Gly Asp                 485 490 495 Gln Val Phe Pro Lys Thr             500  

Claims (9)

A histone deacetylase protein from Brassica rapa ssp. Pekinensis having the amino acid sequence of SEQ ID NO: 6. A gene consisting of a nucleic acid sequence encoding the histone deacetylase protein of claim 1. The gene of claim 2, wherein the nucleic acid sequence has a nucleic acid sequence of SEQ ID NO: 5. 6. Recombinant vector comprising the gene of claim 2. The recombinant vector of claim 4, wherein the recombinant vector is a pB7GWIWG2 (II) -HDI vector shown in FIG. 8. A transformant transformed with the recombinant vector of claim 4. Transgenic cabbage having a plant tumor suppression ability using the transformant of claim 6. (i) preparing the primers set forth in SEQ ID NOS: 1-2; (ii) separating the gene for the histone deacetylase protein by performing RT-PCR on the cDNA synthesized from the RNA of the Chinese cabbage using a primer pair consisting of SEQ ID NOS: 1 to 2; And (iii) identifying the entire sequence of histone deacetylase using the isolated gene. A primer for detecting histone deacetylase protein selected from SEQ ID NOs: 1-2.

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