WO2011002126A1 - Chou chinois transgénique avec tolérance améliorée à la maladie de pourriture molle et son procédé de production - Google Patents

Chou chinois transgénique avec tolérance améliorée à la maladie de pourriture molle et son procédé de production Download PDF

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WO2011002126A1
WO2011002126A1 PCT/KR2009/004440 KR2009004440W WO2011002126A1 WO 2011002126 A1 WO2011002126 A1 WO 2011002126A1 KR 2009004440 W KR2009004440 W KR 2009004440W WO 2011002126 A1 WO2011002126 A1 WO 2011002126A1
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plant
gene
aii
disease
plants
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Yong Pyo Lim
Han Dae Yun
Enkhchimeg Vanjildorj
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The Industry & Academic Cooperation In Chungnam National University
Chungcheongnamdo(Province)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

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  • the present invention relates to a transgenic Chinese cabbage with enhanced tolerance to soft rot disease by expressing aii gene in an apoplast by using a gene encoding PinII signal peptide and aii gene, and production method thereof.
  • Brassica rapa L. ssp. pekinensis (Chinese cabbage) is one of the most important vegetable in Korea, as emphasized by extensive agriculture across 37,200 ha. Chinese cabbage is usually used as the base ingredient of kimchi, the most favorable and common side dish eaten at every Korean meal with rice. A major advantage of consuming Chinese cabbage is the prevention of colon cancer by secondary metabolites, e.g. glucosinolates. Chinese cabbage is a good source of carbohydrates, vitamin A and C, folic acid, and minerals, including: calcium, potassium and iron.
  • Kenshin The Chinese cabbage inbred line, Kenshin, is one of the parental lines of a CKDH mapping population, which was used to construct a reference genetic map for Brassica rapa genome sequencing. To maximize the utility of Kenshin as a reference genetic material for functional genomic research, it is necessary to develop a simple and efficient Agrobacterium -mediated transformation system.
  • Bacterial soft rot is the most severe and destructive disease across members of Brassica .
  • Chinese cabbage is highly susceptible to bacterial soft rot disease, which is caused by a Gram-negative bacterium, Pectobacterium carotovorum subsp . carotovorum ( Pcc ).
  • Pcc Pectobacterium carotovorum subsp . carotovorum
  • B. rapa has the highest soft rot disease severity rating compared to other susceptible species, and the most susceptible B. rapa subspecies are pekinensis and chinensis (Ren et al. 2001b, Euphytica 118: 271-280). Control of soft rot disease is difficult due to a wide range of hosts, the ability of the bacteria to survive in the plant debris in soil, and host susceptibility. Chemical controls are not available.
  • Culturing practices including: raised planting beds, reduced plant density, and delayed planting dates can reduce disease frequency and progression, but may reduce agricultural efficiency (Fritz and Honma, 1987, J Amer Soc Hort Sci 112: 41-44). Therefore, genetic tolerance or resistance may represent an ideal alternative approach. Ren et al. (2001a, Euphytica 117: 197-207) improved soft rot disease resistance of Chinese cabbages through repeated intercrossing. But the traditional recurrent selection procedure requires time- and labor-consuming. Moreover, due to lack of natural resistant gene against soft rot disease, the proceeding of routine raising disease-resistant breeding is restricted (Li 1995, Progress in disease resistant breed of main vegetables. Beijing, Science Press, pp 96-100).
  • Chinese cabbage is generally considered to be recalcitrant in tissue culture (Zhang et al. 1998, Plant Cell Rep 17: 780-786), and there are only few reports on the transformation of hybrid lines (Zhang et al. 2000, Plant Cell Rep 19: 569-575; Zhao et al. 2006, Euphytica 150: 397-406) and some inbred lines (Kim et al. 2003, J Kor Soc Hort Sci 44: 5-9; Min et al. 2007, Plant Cell Rep 26: 337-344). None of these studies involved the transfer of soft rot disease tolerant or resistant genes.
  • autoinducers such as N -acyl homoserine lactones (AHLs) regulate particular virulence gene expression in plants and animals.
  • the autoinducer inactivation gene ( aii ) family members encode AHL inactivation enzymes (e.g. AHL-lactonase) that significantly reduce the release of AHLs, decrease extracellular pectolytic enzyme activities and attenuate pathogenicity of Pcc for potato, eggplant, Chinese cabbage, carrot, celery, and tobacco (Dong et al. 2000, Nature 411: 813-817; 2001, PNAS 97: 3526-3531).
  • Signal peptides may target the recombinant proteins to the apoplast of transgenic tobacco and potato plants (Murray et al. 2002, Transgenic Res 11: 199-214). Therefore, the pin IISP -aii fusion gene may target AHL-lactonase to the intercellular space of Chinese cabbage tissues where Pcc initiates infection.
  • the objective of present study was to introduce the fusion gene ( pin IISP -aii ) into Chinese cabbage inbred line Kenshin genome to produce genetically modified plants capable of quenching pathogen quorum-sensing signaling.
  • the present invention is devised in view of the above-described needs. Specifically, inventors of the present invention found that, when fusion gene pin IISP -aii is introduced to the genome of Chinese cabbage inbred line, Kenshin, for the production of a genetically modified plant which is capable of quenching a quorum-sensing signal, the transgenic plant in which pin IISP -aii is expressed exhibits significantly increased tolerance to soft rot disease compared to the wild-type plant. As a result, the present invention was completed.
  • the present invention provides a recombinant plant expression vector comprising a gene encoding the signal peptide for PinII (protease inhibitor II) from potato and aii (autoinducer inactivation) gene from Bacillus sp. GH02.
  • the present invention provides a plant transformed with the vector described above so as to have improved tolerance to pathogenic disease, and seeds of the plant.
  • the present invention provides a method of improving tolerance of a plant to pathogenic disease comprising steps of transforming a plant with the vector described above and expressing the aii gene in apoplast of the plant.
  • the present invention provides a composition for improving tolerance of a plant to pathogenic disease comprising a gene encoding the signal peptide for PinII from potato and aii gene from Bacillus sp. GH02 as described above.
  • the transgenic Chinese cabbage of the present invention expressing the pin IISP- aii fusion gene can express the aii gene in apoplast of the plant, and therefore the present invention has an excellent effect of providing transgenic Chinese cabbage having improved tolerance to soft rot disease. Therefore, the present invention is very valuable invention in crop industry.
  • Figure 1 shows regeneration of pin IISP- aii transgenic Chinese cabbage inbred line Kenshin via Agrobacterium-mediated transformation.
  • Hygromycin-resistant calli derived on the edges of cotyledon explants after five weeks of selection.
  • Hygromycin-resistant adventitious shoots regenerated within 14 days.
  • Root regenerated within three weeks.
  • Self pollination i
  • FIG. 2 shows schematic diagram of the T-DNA region of the binary vector pCAMBIA1302 ( a ).
  • gfp green fluorescent protein gene
  • SP protease inhibitor II signal peptide from potato
  • aii autoinducer inactivation gene from Bacillus sp. GH02
  • hptII hygromycin phosphotransferase II gene
  • CaMV 35S cauliflower mosaic virus 35S promoter
  • nos3, nopaline synthase transcription terminator RB and LB, right and left borders.
  • b Southern blot analysis of wild-type (WT) and T 0 pin IISP- aii transgenic lines T1 ⁇ T5.
  • FIG 3 shows tissue inoculation with Pectobacterium carotovorum subsp . carotovorum .
  • Gray (WT), white (T101), and dotted (T103) columns indicate the mean of three replicates, and bars represent ⁇ standard deviation, respectively.
  • Figure 4 shows evaluation of tolerance against soft rot disease at seedling stage.
  • the present invention provides a recombinant plant expression vector comprising a gene encoding the signal peptide for PinII (protease inhibitor II) from potato and aii (autoinducer inactivation) gene from Bacillus sp. GH02.
  • the PinII signal peptide recruits the recombinant protein to apoplast of a transgenic plant.
  • it may consist of an amino acid sequence that is represented by SEQ ID NO: 1.
  • the above-described aii gene may consist of a nucleotide sequence that is represented by SEQ ID NO: 2, but not limited thereto.
  • the above-described recombinant plant expression vector can be pCAMBIA1302 vector shown in Fig. 2, for example. However, it is not limited thereto.
  • recombinant indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide.
  • Recombinant cell can express a gene or a gene fragment in a form of a sense or antisense, which is not found in natural state of cell.
  • a recombinant cell can express a gene that is found in natural state, provided that said gene is modified and re-introduced into the cell by an artificial means.
  • vector is used herein to refer DNA fragment(s) and nucleotide molecules that are delivered to a cell.
  • Vector can replicate DNA and be independently reproduced in a host cell.
  • delivery system and “vector” are often interchangeably used.
  • expression vector means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequences that are essential for the expression of the operatively-linked coding sequence in a specific host organism. Promoter, enhancer, termination codon and polyadenylation signal that can be used for a eukaryotic cell are well known in the art.
  • a preferred example of plant expression vector is Ti-plasmid vector which can transfer a part of itself, i.e., so called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens.
  • Other types of Ti-plasmid vector are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a genome of a plant.
  • Especially preferred form of Ti-plasmid vector is a so called binary vector which has been disclosed in EP 0 120 516 B1 and USP No. 4,940,838.
  • vector that can be used for introducing the DNA of the present invention to a host plant can be selected from a double-stranded plant virus (e.g., CaMV), a single-stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector.
  • a double-stranded plant virus e.g., CaMV
  • a single-stranded plant virus e.g., a single-stranded plant virus
  • a viral vector which can be originated from Gemini virus, etc. for example a non-complete plant viral vector.
  • Use of said vector can be advantageous especially when a plant host cannot be appropriately transformed.
  • Expression vector would comprise at least one selective marker.
  • Said selective marker is a nucleotide sequence having a property based on that it can be selected by a common chemical method. Every gene which can be used for the differentiation of transformed cells from non-transformed cell can be a selective marker.
  • Example includes, a gene resistant to herbicide such as glyphosate and phosphinotricin, and a gene resistant to antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.
  • a promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoters, but not limited thereto.
  • the term “promoter” means a DNA molecule to which RNA polymerase binds in order to initiate its transcription, and it corresponds to a DNA region upstream of a structural gene.
  • plant promoter indicates a promoter which can initiate transcription in a plant cell.
  • constitutive promoter indicates a promoter which is active in most of environmental conditions and development states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing a constitutive promoter is not limited herein.
  • any conventional terminator can be used.
  • Example includes nopaline synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens , etc., but are not limited thereto.
  • NOS nopaline synthase
  • rice ⁇ -amylase RAmy1 A terminator a terminator for optopine gene of Agrobacterium tumefaciens , etc.
  • phaseoline terminator a terminator for optopine gene of Agrobacterium tumefaciens , etc.
  • the use of terminator is highly preferable in view of the contexts of the present invention.
  • the present invention further provides a plant transformed with the recombinant plant expression vector of the present invention to have improved tolerance to pathogenic disease.
  • Plant transformation means any method by which DNA is delivered to a plant. Such transformation method does not necessarily need a period for regeneration and/or tissue culture. Transformation of plant species is now quite general not only for dicot plants but also for monocot plants. In principle, any transformation method can be used for introducing a hybrid DNA of the present invention to appropriate progenitor cells. The method can be appropriately selected from a calcium/polyethylene glycol method for protoplasts (Krens et al. 1982, Nature 296, 72-74; Negrutiu et al. 1987, Plant Mol. Biol.
  • a method preferred in the present invention includes Agrobacterium mediated DNA transfer.
  • so-called binary vector technique as disclosed in EP A 120 516 and USP No. 4,940,838 can be preferably adopted for the present invention.
  • the "plant cell” that can be used for the plant transformation in the present invention can be any type of plant cell. It includes a cultured cell, a cultured tissue, a cultured organ or a whole plant.
  • plant tissue can be either differentiated or undifferentiated plant tissue, including root, stem, leaf, pollen, seed, cancerous tissue and cells having various shape that are used for culture, i.e., single cell, protoplast, bud and callus tissue, but not limited thereto. Plant tissue can be in planta or in a state of organ culture, tissue culture or cell culture.
  • the transgenic plant may include not only a whole plant but also a tissue, a cell or a seed which can be obtained from the plant.
  • the plant which is transformed with pin IISP- aii fusion gene of the present invention shows excellent tolerance to pathogenic disease.
  • tolerance of a plant to pathogenic disease means that, even when a plant is attacked by bacteria, filamentous microbes (i.e., fungi), viruses, nematodes, etc., symptom of disease is minor or the plant is not much affected by the disease.
  • the tolerance of a plant to pathogenic disease indicates tolerance of a plant to bacteria, filamentous microbes (i.e., fungi), viruses, nematodes, etc.
  • it indicates tolerance of a plant to bacteria.
  • the above-described bacteria are Pectobacterium carotovorum subsp . carotovorum .
  • the above described disease-causing harmful insect is preferably soft rot disease.
  • the above-described plant is a dicot plant including Arabidopsis thaliana , eggplant, tobacco, pepper, tomato, burdock, crown daisy, lettuce, Chinese bellflower, chard, spinach, sweet potato, celery, carrot, dropwort, parsley, Chinese cabbage, cabbage, leaf mustard radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soy bean, mung bean, kidney bean, sweet pea and the like. More preferably, it is Chinese cabbage ( Brassica rapa L. ssp. pekinensis ).
  • the present invention further provides the seeds of the plant described above.
  • the present invention further provides a method of improving tolerance of a plant to pathogenic disease comprising steps of transforming a plant with the vector described above and expressing the aii gene in apoplast of the plant.
  • a method of overexpressing the aii gene in a plant it can be carried out by introducing the aii gene to a plant which does not comprise the aii gene or to a plant which already comprises the aii gene.
  • the term "overexpressing the aii gene” means that the aii gene is expressed to the level which is higher than normal expression obtained from a wild-type plant.
  • a method of introducing the aii gene to a plant a method of transforming a plant with an expression vector comprising the aii gene, which is under control of a promoter, can be included.
  • a promoter it is not specifically limited if it can be used for the overexpression of a gene which has been introduced to a plant.
  • a promoter include 35S RNA and 19S RNA promoter of CaMV; full-length transcription promoter originating from figwort mosaic virus (FMV) or a promoter for coat protein of TMV.
  • FMV figwort mosaic virus
  • a promoter for coat protein of TMV a promoter for overexpress the aii gene in a monocot plant or a woody plant.
  • an ubiquitin promoter can be used to overexpress the aii gene in a monocot plant or a woody plant.
  • both monocot and dicot plants can be included.
  • Examples of a monocot plant include rice, wheat, barley, bamboo shoot, corn, taro, asparagus, onion, garlic, scallion, leek, wild rocambole, hemp, and ginger, but not limited thereto.
  • Examples of a dicot plant include, tobacco, Arabidopsis thaliana , eggplant, pepper, tomato, burdock, crown daisy, lettuce, Chinese bellflower, chard, spinach, sweet potato, celery, carrot, dropwort, parsley, Chinese cabbage, cabbage, leaf mustard radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soy bean, mung bean, kidney bean, sweet pea and the like, but not limited thereto. More preferably, it is Chinese cabbage ( Brassica rapa L. ssp. pekinensis ).
  • Agrobacterium tumefaciens mediated transformation method As for the method which can be used for delivery of the vector of the present invention to a plant, Agrobacterium tumefaciens mediated transformation method, a microscopic injection method (Capecchi, 1980, Cell 22 : 479), calcium-phosphate method (Graham et al. 1973, Virology 52 : 456), an electroporation method (Neumann et al. 1982, EMBO J. 1 : 841), liposome-mediated transformation (Wong et al. 1980, Gene 10 : 87), DEAE-dextran treatment method (Gopal, 1985, Mol. Cell Biol. 5 : 1188-1190), gene bombardment method (Yang et al. 1990, Proc. Natl. Acad. Sci. 87 : 9568-9572) and the like can be used for introducing the vector to a plant.
  • the present invention provides a composition for improving tolerance of a plant to pathogenic disease, comprising a gene encoding the signal peptide for PinII from potato and aii gene from Bacillus sp. GH02 described above.
  • the PinII signal peptide may preferably consist of an amino acid sequence that is represented by SEQ ID NO: 1 and the above-described aii gene may preferably consist of a nucleotide sequence that is represented by SEQ ID NO: 2.
  • the PinII signal peptide may include the PinII signal peptide wherein insertion, substitution or deletion of a specific amino acid sequence is further comprised.
  • the above-described aii gene may include the aii gene wherein insertion, substitution or deletion of a specific nucleotide sequence is further comprised.
  • Bacillus sp. GH02 was isolated from ginseng root (Cho et al. 2007, Microbial Ecology 54: 341-351) and was cultured at 28 °C.
  • the pGEM-T Easy vector (Promega, WI, USA) was used for cloning and sequencing.
  • pCAMBIA1302 binary vector pCAMBIA1302 (CAMBIA, Australia) which contains the fusion gene ( pin IISP- aii ), signal peptide of protease inhibitor II from potato (PinII) fused to autoinducer inactivation ( aii ) gene (GenBank accession number, FJ189472) from Bacillus sp. GH02, the green fluorescent protein ( gfp ) gene, and a hygromycin phosphotransferase II ( hpt II) gene for resistance to hygromycin (Fig. 2a).
  • fusion gene pin IISP- aii
  • aii signal peptide of protease inhibitor II from potato
  • aii autoinducer inactivation gene
  • gfp green fluorescent protein
  • hpt II hygromycin phosphotransferase II
  • the binary vector was introduced into Agrobacterium tumefaciens LBA4404 by electroporation (Gene Pulser II Electroporator, Bio-Rad, Hercules, CA, USA). A. tumefaciens strains were grown overnight in YEP medium prior to use in transformation.
  • degenerate oligonucleotide primers were designed based on conserved amino acid sequences from highly conserved regions.
  • the sense and antisense degenerate oligonucleotide primers used were 5'- CAC YTR CAT YTT GAY CAY GC -3' (sense, 694F; SEQ ID NO: 3) and 5'- ATC RAA TCC HGA YRA YGG C -3' (antisense, 695R; SEQ ID NO: 4), respectively.
  • PCR amplification using genomic DNA of Bacillus sp. GH02 as a template was carried out using Super-Therm DNA polymerase (JMR, Side Cup, Kent, UK) under 30 cycles of denaturation at 94 °C for 30 sec, annealing at 50 °C for 30 sec, and extension at 72 °C for 30 sec.
  • the amplified product of approximately 300 bp was isolated from an agarose gel using a gel extraction kit (Intron Biotechnology, Seongnam, Korea).
  • the PCR product was sequenced, the sequence was confirmed by BLAST searches. From the sequenced DNA the 5' upstream and 3' downstream regions were amplified by primer walking using only gene specific primers based on the determined sequence.
  • the amplified fragments were isolated for further nucleotide sequencing.
  • the complete open reading frame (ORF) was amplified from genomic DNA using specific primers (705F and 803R) and cloned into the pGEM-T easy vector (Table 1).
  • Reported nucleotide sequence data is available in the GenBank database under the accession number FJ189472 for the aii gene, and DQ365557 for the 16S rDNA of Bacillus sp. GH02.
  • pin IISP aii the potato pin IISP gene and Bacillus sp. GH02 aii gene were amplified from chromosomal DNA with the primer set, pin IISP (1948F-1950R) and aii (1949F-1099R), respectively (Table 1). Overlap sites were introduced into 1949F-1950R.
  • the pin IISP and aii gene fragments were amplified with primers 1948F-1950R and 1949F-1099R, then were purified by gel extraction using a kit (Intron Biotechnology, Seongnam, Korea), ligated into pGEM-T easy vector and were transformed into E. coli DH5 ⁇ .
  • A. tumefaciens stock Five ⁇ l of A. tumefaciens stock was cultured in 5 ml YEP medium containing 100 mg l -1 kanamycin at 28 °C. After 24 h of culture, 10 ⁇ l of aliquot was sub-cultured again in 10 ml YEP medium with 100 mg l -1 kanamycin until an OD 600 value of 0.6. The bacterial suspension was centrifuged at 3500 rpm for 15 min, and the pellet was resuspended in 10 ml of resuspension medium (MS with 50 mg l -1 acetosyringone).
  • the cotyledonary explants (0.3 x 0.3 cm 2 ) were cultured on pre-cultivation medium MSRM (MS with 5 mg l -1 BA, 0.5 mg l -1 NAA , 2 mg l -1 AgNO 3 , 16 g l -1 phytoagar, pH 5.8) in the light for three days.
  • MSRM MS with 5 mg l -1 BA, 0.5 mg l -1 NAA , 2 mg l -1 AgNO 3 , 16 g l -1 phytoagar, pH 5.8
  • the pre-cultured explants were immersed in bacterial suspension for 10 min and then cultured on co-cultivation medium (MSRM with 50 mg l -1 acetosyringone) in the dark at 25 °C for three days.
  • the explants were rinsed three times in sterile distilled water, and once in 500 mg l -1 carbenicillin containing-sterile distilled water for 10 min, and then surface-dried on sterilized filter paper. For selection, the explants were transferred to callus regeneration/selection medium (MSRM with 250 mg l -1 carbenicillin and 10 mg l -1 hygromycin).
  • MSRM callus regeneration/selection medium
  • the adventitious shoots were transferred to rooting medium (1/2 MS with 250 mg l -1 carbenicillin, 10 mg l -1 hygromycin, 8 g l -1 phytoagar).
  • rooting medium 1/2 MS with 250 mg l -1 carbenicillin, 10 mg l -1 hygromycin, 8 g l -1 phytoagar.
  • the rooted plants were transferred to the soil.
  • the hygromycin-resistant plants were transferred to 4 °C and cultured during 45 days for vernalization. Finally, the plants were transferred to greenhouse and then self-pollinated.
  • Genomic DNA was extracted from young leaf tissues using Cetyl trimethylammonium bromide (CTAB) (Saghai-Maroof et al. 1984, PNAS 81: 8014-8018). About 30 ⁇ g of Hin dIII-digested genomic DNA samples were electrophoresed on an 0.8% (w/v) agarose gel, then depurinated in 0.25 N HCl for 10 min, denatured in 0.5 N NaOH and 1.5 N NaCl for 1 h, neutralized in 1 M Tris (pH 7.5) and 1.5 M NaCl for 1h, and washed in 10 x SSC for 1 h.
  • CAB Cetyl trimethylammonium bromide
  • pin IISP -aii gene was amplified using primer set, 5'- TAC TTG TAA GCG CGA TGG AG -3' (forward; SEQ ID NO: 5) and 5'- AGA TGA AGT GCC ATT TGC G -3' (reverse; SEQ ID NO: 6), yielded about 532 bp PCR product.
  • the following PCR conditions were used: 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec (33 cycles). Hybridization was performed overnight at 65 °C in Rapid-hyb buffer (Amersham Pharmacia Biotech, UK). Subsequent steps of Southern and Northern blot analyses were performed as described by Enkhchimeg et al. (2005, Plant Cell Tiss Org Cult 83: 41-50).
  • the data analysis was done with SAS 9.1 (SAS Institute Inc., Cary, NC USA) and was compared by ANOVA (one way) with Duncan's multiple range test ( P > 0.05).
  • T 1 hygromycin-resistant plants of T101 and T103 transgenic lines were transplanted into plastic pods with peat moss and perlite (1:1). The plants were covered by wrap with small holes and grown in the culture room adjusted to 25 ⁇ 1 °C and 18-h photoperiod of 30 ⁇ mol m -2 s -1 illumination provided by cool white fluorescent lamps. Approximately in 10 days of transplantation, the plants at the 5 ⁇ 7-leaf stage were used to evaluate of tolerance against soft rot disease.
  • the plant inoculation was performed as described by Lee and Cha (2001, Phytopath 91: S53-S54). Each plant was drenched by pouring 5 ml of a 4:1 mixture of Pcc and sterile mineral oil (heavy white oil, Sigma) over the center of the plant. The inoculated plants were then covered by wrap with small holes and grown in the culture room as described above. The soil, peat moss and perlite (1:1), was kept with constantly enough moisture.
  • the inoculated plants were examined for soft rot daily after Pcc inoculation.
  • the disease test was performed on 25 plants per replicate and it was repeated three times.
  • the data analysis was done with SAS 9.1 (SAS Institute Inc., Cary, NC USA) and was compared by ANOVA (one way) with Duncan's multiple range test ( P > 0.05).
  • Example 1 Construction of the fusion gene by overlap PCR
  • Kenshin, 4 day-old cotyledon explants were inoculated with A. tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1302 carrying pin IISP -aii , gfp , and hptII genes.
  • a co-cultivation period of two or three days was determined to be optimal.
  • explants of Brassica are very sensitive to co-cultivation with Agrobacterium , and a co-culture extension period caused necrosis during subsequent cultivation.
  • pin IISP -aii transgene Integration of the pin IISP -aii transgene into the genome of transgenic plants was confirmed by Southern blot analysis. A single copy of the transgene was successfully integrated into each of the four transgenic lines (T1 ⁇ T4), and two copy numbers transgene were integrated in one transgenic line (T5), but not in wild-type plant (Fig. 2b). The expression of the pin IISP -aii transgene in the transgenic plants was confirmed by Northern blot analysis. The pin IISP -aii transcript was detected in all the transgenic plants, but not in the wild-type plant (Fig. 2c).
  • Example 4 Evaluation of tolerance against soft rot disease
  • T101 and T103 showing high expression of pin IISP- aii gene were selected for further experiment.
  • disease symptom evaluation was carried out on tissues of detached leaves of wild type, T101 and T103 plants.
  • Significant differences of disease symptom development was observed between wild type plants and transgenic lines at 1 day, 3 days, 6 days and 10 days post inoculations, while two transgenic lines showed significant difference of disease development between them at 6 days and 10 days post inoculations (Fig. 3a).
  • Greater symptoms of disease development in detached leaves of wild type plants were observed from 1 day post inoculation to 10 days post inoculation compared to transgenic lines, thereby suggesting that transgenic plants expressing pin IISP -aii experienced delayed soft rot symptom development.
  • Induction of soft rot disease by artificial inoculation is important for many areas, such as breeding of resistance cultivars, evaluation of pesticides, and study of host-pathogen interaction.
  • an artificial inoculation was carried out by pouring the mixture of mineral oil and bacterial suspension on center of the plants without wounding and keeping them inside the moist chamber, whereas none of the plants showed soft rot symptom by just bacterial suspension.
  • fusion gene pin IISP -aii reduces susceptibility to soft rot disease in Chinese cabbage.
  • Transgenic tolerance must be the result of recombinant quorum-quenching enzyme AHL-lactonase activity since this enzyme is encoded by aii .
  • the enzyme can effectively quench bacterial quorum-sensing signaling and disintegrate bacterial population density-dependent infections.
  • Bacterial soft rot is not only a serious disease for Chinese cabbage, but it often damages all crucifer crops.
  • the pathogen itself is widely distributed in agricultural areas, and in uncultivated land (Kikumoto, 2000, J Gen Plant Pathol 66: 275-277). Pathogen related loss can be massive, either in the field or during vegetable transport and preservation. Therefore, the control of soft rot disease in the Chinese cabbage industry is of high priority.

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Abstract

La présente invention porte sur un vecteur d'expression de plante recombinante comprenant un gène codant pour le peptide signal pour PinII (inhibiteur de protéase II) provenant de la pomme de terre et un gène aii (d'inactivation auto-inducteur) provenant de Bacillus sp. GH02, sur une plante transformée par le vecteur décrit ci-dessus de façon à avoir une tolérance améliorée à une maladie pathogène et sur des graines de la plante, sur un procédé d'amélioration de la tolérance d'une plante à une maladie pathogène comprenant les étapes consistant à transformer une plante par le vecteur décrit ci-dessus et à exprimer le gène aii dans l'apoplaste de la plante, et sur une composition pour améliorer la tolérance d'une plante à une maladie pathogène comprenant un gène codant pour le peptide signal pour PinII provenant de la pomme de terre et un gène aii provenant de Bacillus sp. GH02.
PCT/KR2009/004440 2009-06-30 2009-08-10 Chou chinois transgénique avec tolérance améliorée à la maladie de pourriture molle et son procédé de production WO2011002126A1 (fr)

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KR10-2009-0058795 2009-06-30
KR1020090058795A KR101108971B1 (ko) 2009-06-30 2009-06-30 무름병 내성이 증진된 배추의 형질전환체 및 그 제조방법

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WO2011002126A1 true WO2011002126A1 (fr) 2011-01-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022125894A1 (fr) * 2020-12-10 2022-06-16 Colorado State University Research Foundation Inhibiteurs de protéase et leur utilisation pour fournir une résistance aux maladies dans des plantes et en tant qu'antimicrobiens

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102459931B1 (ko) * 2020-09-18 2022-10-31 한국식품연구원 미산성차아염소산수를 이용한 배추 무름(연부병) 부패균(Pectobacterium carotovorum subsp. carotovorum) 제어용 살균 방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6066491A (en) * 1990-01-30 2000-05-23 Zeneca Mogen B.V. Process for obtaining fungal resistant plants with recombinant polynucleotides encoding β-1,3-glucanase modified for apoplast targeting
US7205452B2 (en) * 2001-01-29 2007-04-17 Agency For Science, Technology And Research Control of bacterial infection by quenching quorum-sensing of plant pathogenic bacteria
KR20070073715A (ko) * 2007-06-26 2007-07-10 중앙대학교 산학협력단 화경조직을 이용한 배추의 형질전환체 제조방법 및이로부터 생산되는 무름병 저항성이 증진된 형질전환체

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6066491A (en) * 1990-01-30 2000-05-23 Zeneca Mogen B.V. Process for obtaining fungal resistant plants with recombinant polynucleotides encoding β-1,3-glucanase modified for apoplast targeting
US7205452B2 (en) * 2001-01-29 2007-04-17 Agency For Science, Technology And Research Control of bacterial infection by quenching quorum-sensing of plant pathogenic bacteria
KR20070073715A (ko) * 2007-06-26 2007-07-10 중앙대학교 산학협력단 화경조직을 이용한 배추의 형질전환체 제조방법 및이로부터 생산되는 무름병 저항성이 증진된 형질전환체

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022125894A1 (fr) * 2020-12-10 2022-06-16 Colorado State University Research Foundation Inhibiteurs de protéase et leur utilisation pour fournir une résistance aux maladies dans des plantes et en tant qu'antimicrobiens

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KR20110001312A (ko) 2011-01-06

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