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    • 专利摘要:
    The present invention relates to an antimicrobial peptide derived from the buds of Chinese cabbage, cDNA encoding the same, a vector for plant transfer in which the cDNA is recombinant, and a transgenic plant having antimicrobial activity prepared using the same. Because of the invention the antimicrobial peptide BSD1 (B rassica s tamen specific plant d efensin-like peptide 1) has a very excellent antibacterial activity, by introducing a gene encoding it to a plant cell, to provide a transgenic plant having an antibacterial activity Can be.
    公开号:KR20030020070A
    申请号:KR1020010052372
    申请日:2001-08-29
    公开日:2003-03-08
    发明作者:이상열；임채오；조무제；박형철；강윤환；김민철；이승식
    申请人:대한민국 (경상대학교 총장)；
    IPC主号:
    专利说明:

    CDNA encoding a novel antifungal peptide, defensin, and disease-resistant transgenic plant prepared by over-expressing the gene}
    [57] The present invention relates to an antimicrobial peptide derived from the buds of Chinese cabbage, cDNA encoding the same, a vector for plant transfer in which the cDNA is recombinant, and a transgenic plant having antimicrobial activity prepared using the same.
    [58] Crops are stuck in constant attack by a number of pathogens (fungus, bacteria, viruses, etc.) and various diseases, resulting in huge yields. The damage of crops caused by these pathogens is not only an important factor that has the greatest impact on agricultural productivity, but also a serious problem called environmental pollution due to pesticides used to improve productivity. To create an environment (Sherman JD et al., Arch. Environ. Health., 52, 332-333, 1997). In addition, farmers spraying pesticides inhale the poisonous pesticides into the human body, creating accumulation and incurable genetic diseases in the human body, and causing fatal health loss for consumers due to absorption of residual pesticides (London et. al., Scan. J., Work.Environ.Health., 24, 18-29, 1998).
    [59] Therefore, researches have been made on methods to prevent the environmental pollution caused by the control of diseases of plants and the use of pesticides or the occurrence of fatal diseases caused by human accumulation. For example, the screening and isolation of pathogenic bactericidal proteins present in living organisms. And cloning of genes encoding them and development of disease-resistant transgenic plants using them. In particular, plants, unlike higher animals, do not have a separate immune system to protect themselves from pathogens, and invade the pathogen by expressing various small antibacterial proteins that are rich in cysteine and glycine. Since they are protecting themselves from the antimicrobial protein of such a small size (2kD ~ 9kD) is being actively researched.
    [60] Antimicrobial proteins of this small size include thionins (Bohlmann et al., Annul. Rev. Plant Physiol. Plant Mol. Biol. 42, 227-240, 1991), plant defensins (Gao et al. , Nature Biotech. 18, 1307-1310, 2000), lipid transfer proteins; Garcia-Olmedo et al., Trends Microbiol. 3, 72-74, 1995), hevein type peptides; Koo et. al., Biochim. Biophys.Acta, 1382, 80-90, 1998), knottin-like peptides; Cammue et al., J. Biol. Chem. 267, 2228-2233, 1992), MBP1 (Duvick et al., J. Biol. Chem. 267, 18814-18820, 1992), 1bMBP (Tailor et al., J. Biol. Chem. 272, 24480-24487, 1997), snakins (Segura et al. , Mol. Plant-Microbe.Interact. 12, 16-23, 1999).
    [61] Of these, plant defensins have 45 to 54 amino acid residues, and although the similarity of amino acid sequence is low depending on the type of plant, in common, eight cysteine residues form four disulfide bonds (Broekaert et. al., Plant Physiol. 108, 1353-1358, 1995), Group I defensins inhibiting the growth of Gram-positive bacteria and fungi, Group II defensins, Gram-positive bacteria and Gram resistant to fungi but not to bacteria It is divided into four groups: group III defensin, gram-positive bacteria, gram-negative bacteria and group IV defensins, which are resistant to both fungi but not fungi (Osborn et al., FEBS Lett. 368, 257-262, 1995, De Samblanx et al., Peptide Res. 9, 262-268, 1996; De Samblanx et al., J. Biol. Chem. 272, 1171-1179, 1997; Segura et al., Mol.Plant-Microbe.Interact. 12, 16-23, 1999).
    [62] Known defensin genes are pea (Chiang et al., Mol. Plant-Microbe.Interact. 4, 324-331, 1991), tobacco (Gu et al., Mol. Gen. Genet. 234, 89-96 , 1992), a gene derived from the baby pole (Epple et al., FEBS Lett, 400, 168-172, 1997), and at least five different defensin genes have been identified in the baby pole, of which pdf 2.3 is the seedling. , Pdf 1.2 has been shown to be expressed not only by fungal pathogens, but also by methyl jasmonate (Epple et al., FEBS Lett. 400, 168-172, 1997). In addition, transgenic tobacco plants expressing antimicrobial protein 1 (Rs-AFP1) isolated from the seeds of radish have been reported to exhibit increased resistance to fungal pathogens (Terras et al., Plant Cell. 7, 573). -588, 1995). Plant defensins such as these have been shown to play an important role in the biological defense action of plants because of their antimicrobial activity and increased resistance from fungi of genetically modified plants.
    [63] Therefore, the development of more plant defensins in addition to the above-mentioned defensins for the prevention of environmental diseases and the occurrence of fatal diseases caused by human accumulation by controlling plant diseases and using pesticides.
    [64] Therefore, the present inventors have also studied to develop a new plant defensin which is expected to play an important role in the biodefense action of the plant, and produced a cDNA library from the buds of Chinese cabbage, from which the antimicrobial peptide BSD1 having strong antibacterial activity Was isolated and cloned into the vector. Subsequently, a transformed plant in which the antimicrobial peptide BSD1 was overexpressed was prepared using Agrobacterium tumenfaciens transformed with this vector, confirming that the plant exhibited excellent pathogen resistance, and completed the present invention.
    [65] It is therefore an object of the present invention to provide a cDNA encoding the antimicrobial peptide BSD1 derived from the buds of cabbage.
    [66] Another object of the present invention is to provide a plant transfer vector comprising the cDNA.
    [67] Still another object of the present invention is to provide a recombinant microorganism transformed with the transfer vector.
    [68] Still another object of the present invention is to provide a transgenic plant having antimicrobial activity by expressing antimicrobial peptide BSD1.
    [1] 1A shows the nucleotide sequence of the antimicrobial peptide BSD1 gene of the present invention and the amino acid sequence deduced therefrom (in single letter below the nucleotide sequence).
    [2] <Description of Drawing Symbols>
    [3] Underlined: Predicted Mature Peptides
    [4] Negative Numbers: Signal Peptides
    [5] Number on the right: Number of nucleotides and amino acids
    [6] Rectangular Box: Predicted Poly-A Signal
    [7] Asterisk: Stop Codon
    [8] Coarse cysteine: forming four disulfide bonds
    [9] One arrowhead: processing site between signal peptide and mature BSD1
    [10] FIG. 1B compares the amino acid sequence of the antimicrobial peptide BSD1 of the present invention with that of other plant defensins. Here, the amino acid sequence is shown without the signal peptide, and the bold indicates the conserved residue. And the parenthesis shows the similarity between BSD1 and defensin.
    [11] <Description of Drawing Symbols>
    [12] FST: flower-specific thionin from tobacco; Gu et al., Mol. Gen. Genet. 234, 89-96, 1992
    [13] PPT; Pistil-specific thionin from petunia; Karunanandaa et al., Plant Mol. Biol. 26, 459-464, 1994
    [14] J1: fruit-specific defensin from bell pepper; Meyer et al., Plant physiol. 112, 615-622, 1996),
    [15] γ-H: γ-hordothionin from barley; Colilla et al., FEBS Lett. 270, 191-194, 1990,
    [16] γ1-P and γ2-P: γ1-purothionin and γ2-purothionin from wheat endosperm; Mendez et al., Eur. J. Biochem. 194, 533-539 , 1990), Rs-AFP1 (AFP1 from radish seeds; Terras et al., Plant Cell 7, 573-588, 1995),
    [17] Pdf 1.2 and Pdf 2.3:Arabidopsis ThalianaPlant defensins fromArabidopsis thaliana; Epple et al., FEBS Lett. 400, 168-172, 1997).
    [18] F: flower, f: fruit, S: seed, St: stamen, Pi, pistil
    [19] Rs: Rosette.
    [20] 1C shows the amino acids commonly present in plant defensins and predicted disulfide bonds.
    [21] Figure 2 shows the Southern blotting result of the BSD1 gene using the whole gene of Chinese cabbage.
    [22] Lane E: Eco R I, lane H: Hin d III, lane B: Bam HI
    [23] Figure 3 shows the expression level of BSD1 transcripts analyzed in each tissue (A) and organs (B), and flower differentiation step (C) of the Chinese cabbage.
    [24] <Description of Drawing Symbols>
    [25] I: RNA isolated from green flower tissue with a bud size of 0.3 cm
    [26] II: RNA Isolated from Green Flower Tissue with Peak Size of 0.8cm
    [27] III: RNA Isolated from Yellow Flower Tissue with Flower Size of 1cm
    [28] IV: RNA isolated from flowering flowers.
    [29] 4A is a map of a vector pBSDM constructed for production of recombinant protein in E. coli. Where Amp R is an ampicillin resistance marker and P tac is a tac promoter.
    [30] 4B is a result of confirming the production of water-soluble recombinant BSD1 in E. coli by electrophoresis (lane 1-6) and Western blot analysis (lane 7).
    [31] Lanes 1-4: Total water soluble protein after 0, 1, 2 and 4 hours of IPTG induction
    [32] Lane 5: GST-BSD1 fusion protein fraction
    [33] Lane 6: purely isolated BSD1 after thrombin hydrolysis
    [34] Lane 7: Recombinant BSD1
    [35] Figure 5 shows the results of measuring the antimicrobial activity of the BSD1 peptide in vitro.
    [36] <Description of Drawing Symbols>
    [37] A is a neuro spokes la greater ryasya Petri dish (petri-dish) to (Neurospora crassa) antifungal and antimicrobial peptides BSD1 co-culture a.
    [38] B is oxy Puja Solarium sports Solarium (Fusarium oxysporium) is a co-culture petri dish fungal and antimicrobial peptides BSD1.
    [39] Disc 1 shows the antimicrobial activity (control) after 100 μl of tertiary distilled water was added.
    [40] Disc 2 shows the antimicrobial activity after adding 100 μg of bovine plasma (BSA) protein.
    [41] Disc 3 shows the antimicrobial activity of 25 ㎍1 BSD1 peptide.
    [42] Disc 4 shows the antimicrobial activity after adding 50 ug of isolated BSD1 peptide.
    [43] Disc 5 shows the antimicrobial activity after decomposing BSD1 peptide by treating 50 ㎍ separated BSD1 peptide with pronase 200mg / ㎖ for 2 hours at 37 ℃.
    [44] Figure 6 shows the antibacterial activity of BSD1 in vitro, which is a growth inhibition curve of fungal spores.
    [45] -■-: Fujium Oxysporidium Spore
    [46] -●-: Neurospora Krysha Spore
    [47] - ▲ -: Sora para pies Saratov seutika (Phytophthora parasitica) spores
    [48] 7A is a vector for preparing a transgenic tobacco to BSD1.
    [49] 7B is Northern blotting result of T 2 transgenic tobacco expressing BSD1, and C is Western blot result.
    [50] <Description of Drawing Symbols>
    [51] W: Wild Tobacco
    [52] vec: tobacco transformed with vector only
    [53] # H-1, # H-5, # H-12, # H-17, # H-21 and # H-22: tobacco transformed with BSD1
    [54] FIG. 7D is a result of examining the disease resistance of the transformed tobacco to phytoprosora parastika .
    [55] I and III: Plants transformed with vectors only
    [56] II and IV: Plants Transformed with BSD1
    [69] Hereinafter, the present invention will be described in more detail.
    [70] CDNA of the cabbage-derived antimicrobial peptides of the invention BSD1 (B rassica s tamen specific plant efensin d-like peptide 1) is a base sequence of the N- terminal randomly selected by the obtained clones using a cDNA library prepared from the buds of Chinese cabbage Based on the analyzed sequence, cDNA showing similarity to known plant defensins was selected, and cDNA including the entire BSD1 base sequence was isolated using a partial base sequence as a probe. The gene fragment contained in the cDNA of this BSD1 has a sequence of SEQ ID NO: 1 and has a size of 510 bp. The amino acid sequence of the antimicrobial peptide BSD1 deduced therefrom is shown in SEQ ID NO: 2.
    [71] The present invention also provides a plant transfer vector comprising a cDNA of BSD1 that can be used to prepare a transgenic plant resistant to plant pathogens and a transformed microorganism transformed with the vector. As a preferred embodiment of the present invention, the cDNA fragment of BSD1 was cloned into the XbaI / ClaI region of the binary vector pGA643 to prepare a recombinant plant transfer vector pGA643-BSD1. Subsequently, the agrobacterium tumefaciens EHA101 was transformed with the recombinant vector pGA643-BSD1 to obtain a transformed microorganism which can be used as a medium in the production of a transgenic plant having antimicrobial activity. BSD1 was introduced into the plant to prepare a transgenic plant.
    [72] Subsequently, as an example of a transforming plant having an antimicrobial activity expressing the BSD1 peptide of the present invention, BSD1 cDNA is introduced into tobacco leaf cells using the Agrobacterium tumefaciens in a leaf-engineered transformation method, and the plant adult Cultured under conditions that induce growth, a transgenic tobacco expressing BSD1 was prepared. To measure the resistance to plant pathogens in transgenic plants, infected with the transgenic tobacco blood Saratov Torah Paris sinika (phytphtora parasitica var nitotianae) and the incubation result, transformed only infected 5 days after vector smoke began to appear disease symptoms but to completely kill the infection after 10 days, in the case of transgenic tobacco BSD1 did not show symptoms of a blood infection sinika Saratov Torah Paris. These results suggest that by introducing the gene encoding BSD1 of the present invention into the desired plant cells according to the general method for producing a transgenic plant, it is possible to effectively multiply the antimicrobial properties of various plants including crops.
    [73] Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to these examples.
    [74] Example 1 Cloning of BSD1 cDNA
    [75] 1-1. Preparation of Bud cDNA Library of Chinese Cabbage
    [76] Double stranded cDNA was isolated from total RNA obtained from buds of Chinese cabbage according to Gubler and Hoffman's method (Gubler and Hoffman, Gene, 23, 263-271, 1983), and oligo (dt) primed poly ( A) Reverse transcriptase was made by reacting mRNA with reverse transcriptase obtained from avian myeloblastosis virus and then treated with RNase H and DNA polymerase I. The DNA thus prepared was blunt-ended with T4 DNA polymerase, methylated with Eco RI methylase, digested with Eco RI enzyme, and size-fractionated using Sepharose CL-4B column. CDNA libraries of Chinese cabbage buds were prepared by ligation of more than 2 kb with Eco R1-cleaved λgt11.
    [77] 1-2. Screening of BSD1 cDNA Clone
    [78] A fragment of the BSD1 cDNA was labeled with 32 P [ATP] and was used as a probe to select full-length BSD1 clones from the cDNA library prepared above. Two positive clones were obtained from 5x10 5 cDNA plaques, and this procedure was tested according to the methods of Sambrook and Russell (Molecular cloning, Chapter 11, 2001, Cold Spring Harbour).
    [79] The cDNA of BSD1 was extracted from the clone obtained by the above-mentioned method, and automated base was determined according to Sanger et al., Proc. Natl. Acad. Sci., 74, 5463-5467, 1977. The base sequence was analyzed using a sequencer (Automatic DNA Sequencer, Applied Biosystems model 373A). The analyzed nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of BSD1 deduced therefrom is shown in SEQ ID NO: 2.
    [80] 1-3. Comparison of Amino Acid Sequences with BSD1 and Other Plant Defensins
    [81] The amino acid sequence of the antimicrobial peptide BSD1 isolated from Chinese cabbage (SEQ ID NO: 2) was compared with the amino acid sequence of known plant defensins, and the results are shown in FIGS.
    [82] The deduced amino acid sequence of BSD1 of the present invention has 29 predictable signal peptides at the N-terminus, the predicted cleavage site has a small amino acid at the amino acid residue of -1 (G),- The residue at position 3 (V) was shown to follow well the von Heijne's law (von Heijne, G. Nucl. Acid. Res. 14, 4683-4690, 1986), which has a large non-directional, polar amino acid. Based on the inferred amino acid sequence, the BSD1 protein was expected to have a molecular weight of about 6 kD and appeared to have an isoelectric point of 10.57.
    [83] Overall comparison of BSD1 and other plant defensins showed 27% similarity to J1 (J1 from bell pepper; Meyer et al., Plant Physiol. 112,615-622, 1996), a fruit-specific defensin derived from bell pepper. Low similarity with other plant defensins, including 38% similarity with FST from tobacco; Gu et al., Mol. Gen. Genet. 234, 89-96, 1992). Indicated. Furthermore, compared with Rs-AFP1, a seed-derived defensin of radish, two glycine in positions 13 and 34 and aromatic residues in position 11, which are well conserved in all plant defensins, are lysine in BSD1. (lysine), alanine (alanine), and glycine (glycine) (see Fig. 1 B). However, the location and presence of 8 cysteines and glutamic acid at residue 29, which are believed to form four disulfide bonds, were exactly consistent with the other plant defensins (see FIG. 1C). Thus, although BSD1 of the present invention differs slightly in amino acid sequence compared to other plant defensins, the overall structure of BSD1 can be classified into plant defensin groups.
    [84] In order to determine the number of genes with similarity to BSD1 in the genome of Chinese cabbage, genomic southern blotting was performed using BSD1 cDNA labeled with radioisotopes as a probe ( 2).
    [85] From these results, it was found that there are several isotypes of BSD1 homologes in the genome of cabbage. Thus, the BSD1 and similar proteins encoded by multi-copy genes are found in plants. It can be seen that it performs a very important function in performing the disease resistance function.
    [86] Example 2 Comparison of Expression Patterns of BSD1
    [87] In order to compare and observe the expression patterns of the BSD1 gene isolated in Example 1, Northern blotting was performed for various tissues and organs isolated from cabbage. Northern blot analysis method is as follows.
    [88] 20 μg of each of the leaves, stems, roots and buds of RNA was separated from a 1.5% formaldehyde-agarose gel and then transferred to a nylon membrane. The membrane was exposed to ultraviolet light (UV) and then labeled with radioisotopes at 65 ° C. in a solution of 0.5 M Na 2 HPO 4 (pH 7.2), 1 mM EDTA, 1% BSA) and 7% SDS. The BSD1 probe was added and reacted for one day. The membrane was washed twice for 10 minutes at 37 ° C. in 2XSSC containing 0.1% SDS (1XSSC is 0.15M NaCl and 0.015M sodium citrate), and again 0.1% SDS was removed. It was washed twice for 10 minutes under the condition of 60 ℃ in a 0.2X SSC solution containing. This membrane was dried and exposed to X-ray film at -70 ° C to obtain FIG. 3.
    [89] From Figure 3, it can be seen that the transcripts of BSD1 were expressed only in buds and not in other tissues (see A in Figure 3). In addition, when observing the expression of transcripts for each organ of the flower, it can be seen that only expressed in the surgery, not in other organs (see FIG. 3B).
    [90] Therefore, as mentioned above, the cDNA encoding the protein of the invention can be confident that BSD1 (B rassica s tamen specific plant d efensin-like peptide 1).
    [91] In addition, the flowering process of the cabbage flower was divided into four steps (step I; green buds of 0.3 cm: step II; green buds of 0.8 cm: step III; yellow buds of 1 cm: and step IV; blooming flowers). Next, RNA in each step was isolated and Northern blot analysis was performed in the same manner as above to observe the expression pattern of BSD1 gene.
    [92] As a result, it was observed that the transcript of BSD1 was not observed in step I, the most transcript was expressed in step II, and the transcript gradually decreased until the flowers were fully bloomed (see FIG. 3C). From this fact, most plant defensin genes have been identified as seeds (Bloch and Richardson, FEBS Lett. 279, 101-104, 1991; Terras et al., J. Biol. Chem. 267, 15301-15309, 1992; Terras et al. , Plant Cell, 7, 573-588, 1995; Osborn et al., FEBS Lett. 368, 257-262, 1995; Nitti et al., Eur. J. Biochem. 228, 250-256, 1995), pea Leaves (Ching et al., Mol. Plant-Microbe.Interact. 4, 324-331, 1991), tubers of potatoes (Moreno et al., Eur. J. Biochem. 223, 135-139, 1994), Bell Unlike those expressed in the ripe fruits of bell pepper (Meyer et al., Plant Physiol. 112, 615-622, 1996), BSD1 of the present invention is expressed only in the early stages of plant surgery and flower differentiation. As the defensins to be, it can be seen that the defensins were first invented in the present invention.
    [93] In addition, to observe whether the BSD1 gene is expressed by environmental stimuli, 1 mM salicylic acid, 0.1 mM methyl jasmonate, and 2 mM ethylene ethephon, 2 mM hydrogen peroxide (H 2 O 2 ) and 1 mM silver nitrate (AgNO 3 ) were treated to observe the amount of transcript. As a result, no expression was observed. Thus, it can be seen that the BSD1 gene is a gene that is not expressed by environmental stimuli.
    [94] Example 3 Expression of Recombinant Protein and its Antimicrobial Activity in Escherichia Coli
    [95] 3-1. Expression of Recombinant Protein
    [96] A slightly modified pAK-SS vector (Amersham Pharmacia Biotech.) Used as a protein expression vector in E. coli was used to make proteins synthesized with glutathione-S-transferase (GST).
    [97] That is, a sense primer (5′-AAACCATGGGGTCATGCAAGAGGCAACC-3 ′) and an antisense primer (subsequent to subtracting a putative N-terminal signal sequence predicted from the cDNA of BSD1 of Example 1) primer: 5′-AATTAACCCTCACTAAAGGG-3´) was used to generate Nco I restriction enzyme sites by PCR (polymerase chain reaction). Filling a base sequence of the PCR product with La kinase T4 DNA polymerase, cut with Xho I restriction endonuclease, then cut with restriction enzymes of the Nco I, connects the seat of pAK-SS vector Nco I and Sma I restriction enzymes, the tac promoter Under control, a pBSDM vector including the BSD1 gene under the GST gene was constructed (see FIG. 4A). The nucleotide sequence of the gene was confirmed using an automated DNA sequencer (Applied Biosystems model 373A).
    [98] The recombinant vector pBSDM was introduced into Escherichia coli (BL21 [Lys S] DE3) by electroporation, and then used to clone the BSD1 gene after separating plasmids from E. coli into which the BSD1 gene was introduced. Enzymatic fragmentation was performed, and the fragmented gene was identified by 1% agarose electrophoresis to confirm the introduction of the BSD1 gene (Yun et al, Plant Physiol., 111; 1219-1225, 1996). It was confirmed.
    [99] Transformed Escherichia coli prepared in this manner was incubated in a medium containing an antibiotic of ampicillin (50 μg / ml) and chloramphenicol (33 μg / ml) under conditions of 28 ° C. When the OD 600 of E. coli was about 0.6, 0.4 mM of IPTG (isoprophyl β-D-thiogalactopyranoside) was added to induce the synthesis of recombinant protein.
    [100] The E. coli was incubated at 28 ° C. for 4 hours and then harvested by centrifugation. PBS buffer containing 0.1 mM PMSF (phosphate-buffered saline, containing 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 and 1.8 mM KH 2 PO 4 , pH 7.6), then crushed with an ultrasonic sonicator, and the aqueous fraction was centrifuged at 10,000 g for 20 minutes. The recombinant protein was isolated by glutathione affinity chromatography, and the N-terminus of the recombinant protein was removed by thrombin cleavage to purely separate the BSD1 protein.
    [101] The purified protein was confirmed by SDS-polyacrylamide gel electrophoresis, and the results are shown in B of FIG. 4. In FIG. 4B, lanes 1 to 4 are total water-soluble proteins before the IPTG addition, after 1 hour, after 2 hours, and after 4 hours, lane 4 is a fraction of a GST-BSD1 fusion protein, and lane 5 is after thrombin hydrolysis. , Purely isolated recombinant BSD1. Lane 7, on the other hand, was a Western blot analysis of recombinant BSD1 with an antibody of GST-BSD1.
    [102] From the above results, it can be seen that the protein expressed in transformed Escherichia coli is a protein having a molecular weight of about 6 kDa.
    [103] 3-2. Antimicrobial Activity of Purely Isolated Recombinant Protein
    [104] The separated protein was dialyzed sufficiently at 4 ° C. for 24 hours using 10 liters of tertiary distilled water, and the dialyzed protein solution was dried using a speed-vac apparatus, and then distilled water was added again. The protein solution was prepared at a concentration having an optimum antimicrobial activity.
    [105] Then, the mold-growth inhibiting irradiation (radial growth-inhibition assays;. . Yun et al, Plant physiol 111, 1219-1225, 1996) as Puja Solarium oxy sports Solarium (Fusarium oxysporium) and Neuro spokes la greater ryasya (Neurospora crassa) The antimicrobial activity was measured using, and the results are shown in FIG. 5. From Figure 5, Fujairah Leeum Leeum oxy Spokane and Spokane neuro-called greater of fungus ryasya they could see significantly inhibited the growth by BSD1 protein.
    [106] Microspectrophotometry assays (Terras et al., J. Biol. Chem. 267, 15301-15309, 1992; Hu and Reddy, Plant Mol. Biol. 34, 949-959, 1997) The protein concentration (IC 50 ) required for 50% fungal growth inhibition was measured. That is, Puja Solarium oxy sports Solarium, neuro spokes easier ryasya pie Saratov seashell para seutika each spore suspension (2 × 10 4 / ㎖) shown in the 25 ℃ protein (potato dextrose solution used in the protein of 50㎕ + 50㎕ Broth: Mold growth was measured to a value at 490 nm with an Enzyme-linkes immunosorbent assay (ELISA) plater reader while incubating in a micro-titer plate for 26-44 hours with Difco). . The results are shown in FIG.
    [107] From Figure 6, Puja Solarium oxy sports Solarium, neuro spokes la greater BSD1 protein to inhibit 50% growth of the three molds of ryasya and pie Saratov seashell para seutika This it showed that required approximately 30 ~ 100㎍.
    [108] Example 4 Preparation of BSD1 Plant Transfer Vector and Transgenic Microorganism
    [109] In order to prepare a plant transfer vector incorporating the BSD1 gene, a 510 bp cDNA fragment of BSD1 was used as the binary vector pGA643 (An, G et al, 1988. Plant Mol. Biol. Manuals, Kluwer, Dordrecht, p. A3 / 1-A3). / 19, 1988) was cloned into the Xbal I / Cla I site to produce a recombinant plant transfer vector "pGA643-BSD1" (see Fig. 7A). In Fig. 7A, npt II is a chimeric gene showing kanamycin resistance, which includes a promoter and transcription terminator of a nopaline synthase gene and a neomycin phosphatase II coding sequence, and CaMV is a cauliflower mosaic virus. (cauliflower mosaic virus, CaMV) 35S promoter.
    [110] Subsequently, the recombinant vector pGA643-BSD1 Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens) EHA101 (Clontech, USA) in which the microorganism transformed with an electric shock method (electrophoration) Next, 10㎍ / ㎖ of kanamycin and 5㎍ / ㎖ LB agar plates containing tetracycline were plated, and cultured at 28 ° C. for 2 days to select transformed Agrobacterium tumefaciens .
    [111] Example 5 Preparation of Transgenic Tobacco
    [112] Agrobacterium-mediated leaf fragment transformation (Horsch et al., Science, 227, 1229-1231, 1985) produced transgenic tobacco expressing BSD1.
    [113] That is, the leaves of tobacco cultured in aseptic state were made into disc-shaped sections, and the transformed Agrobacterium tumefaciens cultured appropriately (OD 600 = 0.5) in a 28 ° C shake incubator was co-cultured with a leaf disc (co -cultivation). Co-cultivation is to put the leaf disc in the co-culture medium, add the cultured Agrobacterium tumefaciens , and then incubate for an appropriate time in a 28 ℃ incubator. After co-culture, all leaf discs were transferred to MS 10 medium (Kanamycin 100mg / l, carbenicillin 500mg / l) to induce shoots. Subcultured with fresh MS 10 medium every three weeks. Sections of tobacco cocultured with Agrobacterium tumefaciens by inducing an adventitious shoot were carried out on a medium containing auxin and cytokinin at appropriate concentrations, avoiding callus formation. This medium uses a medium to which antibiotics are added so that only the transformed shoots are differentiated. Subculture every two weeks to induce differentiation of shoots, grow to the appropriate size and transfer to root differentiation medium. In addition, the composition of the MS medium was cut in half, the growth regulator was removed, and low levels of auxin (0.5 M NAA) were added to generate roots from the cultured stems or the targets. In order to purify the transgenic tobacco plants which differentiated the roots in the cabin to the new environmental conditions of the soil, the plants were first taken out of the cabin, and the agar was removed and transferred to the sterilized soil. Plants transferred to the soil gradually grow in the external environment while growing in a closed space with high humidity. At this time, the Agrobacterium tumefaciens that may remain in the plant tissue was removed by washing several times in sterile distilled water containing antibiotics before moving the plant in the soil. In this way a transformed tobacco was prepared.
    [114] On the other hand, individuals of the T 2 generation of homozygotes expressing a large amount of transcripts in selected transgenic tobaccos were selected for antimicrobial activity investigation (FIG. 7B).
    [115] Example 6 Expression and Antimicrobial Activity of BSD1 in Transgenic Tobacco
    [116] 6-1. Immune Blot Analysis
    [117] Each tissue of the transformed tobacco plant of Example 5 was taken, and the sample was prepared with a protease inhibitor cocktail (protease inhibitor cocktail; 100 mg / ml PMSF, 19 mg / ml aprotinin and 25 mg / ml lupetin). (leupeptin), Boehringer Mannheim, GmbH, Germany) was treated with 1: 4 (w / v) to extract the protein solution (homogenization) and centrifuged at 6000 xg to remove the cell fragments. After the supernatant was obtained, 80% saturated ammonium sulfate was added to soak the protein and then protein precipitate was obtained. The protein precipitate was dissolved using a small amount of the protease inhibitor mixture used previously, and dialyzed using 10 L of the same solution. The dialysis protein solution was quantified using Bradford reagent, the proteins were separated by SDS-electrophoresis, and then the proteins separated by 18% electro-transfer. It was transferred to PVDF (Millipore) membrane and reacted with anti-BSD1 antibody (Lee et al., J. Biol. Chem. 270, 21806-21812, 1995). Color reaction was performed with the ECL detection solution to confirm that BSD1 protein was expressed in the transgenic tobacco of the present invention (FIG. 7C).
    [118] Meanwhile, the anti-BSD1 antibody preparation described above was completely mixed with 200 µg of purely isolated GST-BSD1 protein with a hybridization solution (Freund's complete adjuvant), and then, 150 µg of protein was applied to rabbit subcutaneous tissue at intervals of two weeks. It is prepared by injecting the hybridization solution after mixing.
    [119] 6-2. Investigation of Antimicrobial Activity of Transgenic Tobacco
    [120] In 6-1, it was confirmed that the transformed tobacco of the present invention expressed the BSD1 peptide having antimicrobial activity. The antimicrobial activity of the transformed tobacco was examined by the following method. That is, the resistance to the fungus strain (Phytphora parasiticavar nitotianae) was examined using the transgenic individual selected in Example 5 (H vs et al., Proc. Natl. Acad. Sci. USA 96, 766-771, 1999)
    [121] That is, transgenic tobacco was grown in a sterile culture bottle at 25 ° C. for 1 month on MS agar medium. Two agar plugs (1 cm diameter) containing a fungal mycelium (Phytphora parasiticavar nitotianae (ATCC)) are placed at the same distance from the plants in the culture bottle and the control plants transformed with the pGA643 vector alone and the general tobacco plants as controls. The disease resistance of the fungus was examined using several specimens, according to Alexander et al., Proc. Natl. Acad. Sci., USA, 90; 7327-7331, 1993.
    [122] As a result, the disease symptom began to appear in the control tobacco transformed with the vector only after 5 days of infection, but no apparent change was observed in the transformed tobacco in which BSD1 was introduced. After 8 days of infection, the vector-transformed tobacco showed severe disease symptoms such as leaf wilting and stem receding. After 10 days of infection, the entire plant was completely killed by the pathogen. (See DI in FIG. 7). However, in transgenic tobacco in which the BSD1 gene was introduced, it was confirmed that the leaves lived in a fairly healthy state without significant disease symptoms except that only the leaf surface of the cotton in contact with the infected mold was changed yellow (see D-II of FIG. 7). ).
    [123] On the other hand, in order to observe the growth of the fungi in plants of tobacco transformed with the vector and tobacco transformed with BSD1, a fluorescence microscope having a 365 nm exciter and a 420 nm fluorescent filter (fluoresence filter) ( Observed by aniline blue dyeing using a fluoresence microscope; Carl Zeiss, Thornwood, NY). As a result, the tobacco transformed with the vector alone had a complete growth of fungal hyphae (see D-III in FIG. 7), and the tobacco transformed with BSD1 showed the stage of gradually starting to grow mycelial growth, thus ensuring the growth of pathogens. It can be seen that the inhibition (see D-IV in Fig. 7).
    [124] As described above, the use of the cabbage BSD1 gene, which is a peptide having superior pathogenic bactericidal power or pathogen growth ability than low molecular weight bactericidal peptides present in other plants, can be used to prepare transgenic crops resistant to pathogens. It can also be produced as a pathogen-controlling protein, so it can also be used as a biological pollution-free pathogen control material with no toxicity. Therefore, it will be possible to develop a non-toxic biological disease-resistant transgenic crop that can prevent environmental pollution or human diseases by replacing the previously toxic pesticides used for disease control of crops.
    权利要求:
    Claims (6)
    [1" claim-type="Currently amended] Antimicrobial peptides having the nucleotide sequence of SEQ ID NO: 1 derived from cabbage buds BSD1 (B rassica s tamen specific plant efensin d-like peptide 1) the coding cDNA to.
    [2" claim-type="Currently amended] Antimicrobial peptide BSD1 having the amino acid sequence of SEQ ID NO: 2.
    [3" claim-type="Currently amended] Plant transfer vector comprising the cDNA of the antimicrobial peptide BSD1 of claim 1.
    [4" claim-type="Currently amended] I claim 3 transformed with vector using Agrobacterium Tome Pacific Enschede plants (Agrobacterium tumefaciens).
    [5" claim-type="Currently amended] A transgenic plant transformed with Agrobacterium tumefaciens of claim 4 to express BSD1.
    [6" claim-type="Currently amended] The transgenic plant of claim 5, wherein the plant is tobacco.
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    同族专利:
    公开号 | 公开日
    KR100455745B1|2004-11-10|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-08-29|Application filed by 대한민국 (경상대학교 총장)
    2001-08-29|Priority to KR10-2001-0052372A
    2003-03-08|Publication of KR20030020070A
    2004-11-10|Application granted
    2004-11-10|Publication of KR100455745B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2001-0052372A|KR100455745B1|2001-08-29|2001-08-29|A cDNA encoding a novel antifungal peptide, defensin, and disease-resistant transgenic plant prepared by over-expressing the gene|
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