WO2002000837A2 - Nouveau tabac transgenique restreignant l'activite de ses substances nocives - Google Patents

Nouveau tabac transgenique restreignant l'activite de ses substances nocives Download PDF

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Publication number
WO2002000837A2
WO2002000837A2 PCT/KR2001/001113 KR0101113W WO0200837A2 WO 2002000837 A2 WO2002000837 A2 WO 2002000837A2 KR 0101113 W KR0101113 W KR 0101113W WO 0200837 A2 WO0200837 A2 WO 0200837A2
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Prior art keywords
tobacco
vistis
resveratrol
agrobacterium
gene
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PCT/KR2001/001113
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English (en)
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WO2002000837A3 (fr
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Jae-Ho Pyee
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Priority claimed from KR10-2001-0036939A external-priority patent/KR100423072B1/ko
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Priority to AU2001269546A priority Critical patent/AU2001269546A1/en
Publication of WO2002000837A2 publication Critical patent/WO2002000837A2/fr
Publication of WO2002000837A3 publication Critical patent/WO2002000837A3/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/8286—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 insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a product of novel transgenic tobacco, which restrains negative activities of harmful materials of tobacco by using resveratrol biosynthesis gene of Vistis vinifera X Vistis labrusca L. cv. Kyoho. More specifically the present invention relates to a new product of novel
  • This product is produced by inserting the resveratrol biosynthesis gene of Vistis vinifera X Vistis labrusca L. cv. Kyoho. to pBI 121 vector, .that has been known to have anti-stress and anti-cancer effects , thus constructing a recombinant vector(pBIstsyvl), which is again introduced into
  • Resveratrol and it's Polymer Viniferin have been known as phytoalexin in plants, which is a blocking agent that inhibits the growth of penetrated molds from outside.
  • Resveratrol(or pseudostilbene) is produced from a very few plants and so far Polygonum cuspidatum, Eucalyptus, Spruce, Scottish pine, grapes, Arachis hypogaea, lilly ect are known to belong in these
  • Tobacco contains several thousands of chemical substances. More than 200 substances of them are harmful to the human body and cause diseases or worsen the disease including lung cancer, angina pectoris, cerebral infarction, pulmonary emphysema, bronchitis, asthma, stomach ulcer, arteriosclerosis, myocardinal infarction and optical defect. Moreover, the harmful activities of tobacco are due to toxic substances in the tobacco smoke like carcinogens such as nicotine, nitiosamine and benzypyrene. Nicotine affects the lipid metabolism through autonomic nerve system and promotes deposition of cholesterol in arterial wall by decreasing HDL in blood to cause arteriosclerosis. Nicotine also increases the ability of coagulation of blood platelets when blood coagulates, thus causing blood coagulation more easy.
  • the present invention was performed based on the above facts to produce a novel transgenic tobacco which can neutralize harmful substances of tobacco and decrease it's effect remarkably by transforming Vistis vinifera X Vistis labrusca L. cv. Kyoho resveratrol biosynthesis gene extracted as above which has anti-mutation and anti-cancer effects in tobacco.
  • the present product is the novel .transgenic tobacco produced on the basis of new techniques of gene cloning and transformed plants, that have been developed for last five years and has an excellent effect on anti-cancer and anti-oxidation.
  • the present invention uses the plant breeding gene that has the functions of the original anti-biotic and anti-virus effect so that damages of insects occurring in cultivating tobacco can be reduced and also applied to harvest better quality of tobacco in a large amount. Consequently, the object of the present invention is to provide a constructed recombinant vector by inserting the resveratrol biosynthesis gene of Vistis vinifera X Vistis labrusca L. cv. Kyoho resveratrol in the vector. Another object of the present invention is to provide a transformed Agrobacterium strain (pBIstsyv/LBA4404) obtained by the above recombinant vector introducing into Agrobacterium- LBA4404. Still another object of the present invention is to provide a novel transgenic tobacco that has an effect on neutralizing tobacco toxin substance effectively by inoculating the above strain into tobacco. Disclosure of invention
  • the object of the present invention is to construct a recombinant vector (pBIstsyvl) by inserting the stsy cDNA whose open reading frame and partially untranslated region was digested with a restriction enzyme into the downstream of the CaMV promoter of the pB1121 vector which the GUS gene had been eliminated.
  • the stsy cDNA was amplified by PCR using the first strand cDNA as a primer that was synthesized from the total RNA, which was extracted from each steps of developing tissues and organs of Vistis vinifera X Vistis labrusca L. cv. Kyoho.
  • the leaf explant of the tobacco was transformed by inoculating on above transformed Agrobacterium strain suspension and was cultured on antibiotic added medium. The grown shoots from the medium was rooted and graft- taken to produce a novel transgenic tobacco which restrains toxic substance activity.
  • FIG 1. is showing the nucleotide sequence of stilbene synthase cDNA of Vistis vinifera X Vistis labrusca L. cv. Kyoho.
  • FIG 2. is a graph showing the present invention of the recombinant vector(pBIstsyvl) that carries the gene which synthesize the de-toxicating substance and which is used for transformation.
  • FIG 3. is showing the wild type species of the tobacco and the novel transgenic tobacco which was inserted by FIG 1 gene.
  • FIG 4. is showing the RT-PCR results that shows the expression of de- toxicating substance gene of the novel transgenic tobacco.
  • FIG 5. is showing the analyzed results of the detoxicating substance production yield of novel transgenic tobacco by HPLC.
  • the present invention is executed, comprising the steps of: constructing the recombinant vector by inserting the PCR amplified resveratrol biosynthesis gene in pBI 121 vector; selecting cultured Agrobacterium-LBA4404 as a strain for transformation and introducing the recombinant vector which has resveratrol biosynthesis gene into the strain; moculating the leaf explant with the transformed Agrobacterium; producing a novel transgenic tobacco restiaining toxic activity of tobacco by growing shoots and rooting of the transformed leaf explant in the antibiotic added medium; confirming the resveratrol gene introduction and expression by RT- PCR after isolating total RNA from the leaf of transgenic novel tobacco and isolating genomic DNA followed by genomic PCR; and analyzing the resveratrol content of the transgenic tobacco by HPLC.
  • Example 1 A recombinant vector inserted by the resveratrol gene of Vistis vinifera X Vistis labrusca L. cv. Kyoho and the transformed Agrobacterium introduced by the above recombinant vector.
  • Step 1 RNA and genomic DNA isolation of each organ and tissue of Vistis vinifera X Vistis labrusca L. cv. Kyoho for the production of novel transgenic tobacco.
  • Vistis vinifera X Vistis labrusca L. cv. Kyoho was harvested at every week for 15 weeks after blooming. 5 grapes from the harvested ones were chosen randomly to investigate the fruit development stage with checking the length, width and weight of the fruit.
  • RNA extraction buffer was mixed with the RNA extraction buffer and was centrifuged for 15 minutes at 200 X g in a syringe, which was filled with glass wool. The extract was harvested and precipitated with 2.5 volumes of ethanol. The pellet was dissolved in lOmM Tris-HCl, lmM EDTA, 0.2% ⁇ -mercaptoethanol and then extracted with phenol- chloroform. The supernatant was collected and the RNA was precipitated with 0.1 volume of 3M sodium acetate and 2.5 volume of ethanol.
  • RNA was measured by UV spectrophotometer and RNA gel.
  • isolating genomic DNA 2g of young leaf tissue was grinded by using liquid nitrogen and resuspended with 25ml of buffer A followed by centrifugation at 5000 rpm for 10 minutes. 5ml of extraction buffer was added to the pellet and was incubated at 37 for 30 minutes at a rotary shaker.
  • Step 2 Amplifying cDNA by RT-PCR using primers.
  • Isolated total RNA from each organs and tissues were used for synthesizing the first strand cDNA by using first-strand cDNA synthesis kit
  • the primer which is to amplify the first strand cDNA by PCR.
  • HPPN 5 'ATGGCTTCAGTCGAGGAAATT 3 ' (2 lmer)
  • HPPC HPPC ; 5' TTAATTTGTCACCATAGGAATG 3' (22mer) PCR conditions were decided according to the procedure of Sambrook et al.
  • Step3 Sequencing and analyzing the above cDNA
  • the above RT-PCR amplified cDNA product was serial deleted or subcloned into pCR2.1-TOPO cloning vector several times and the sequence were determined by dideoxy termination method using sequenase kit. After translating the nucleotide into amino acids, the homology between cDNA and the well-known stilbene synthase gene, was analyzed. As a result, it had over 99% homology with the Shiraz stilbene synthase cDNA sequence. In addition, this cDNA was used as a probe for genomic southern hybridization. For analysis, DNASIS/PROSI program was used.
  • Step 4 Culturing Agrobacterium as a strain for Transformation
  • Agrobacterium-LBA4404 for transformation, it was incubated at 28 ° C for 2-3 days after streaking on a YEP solid medium(An, 1987) added rifampicin lOOmg/L, kanamycin(Km) lOOmg/L, agar 15g/L. After picking a colony and inoculating 3ml of YEP broth containing Km lOOmg/L, it was incubated at 28 for 8 hours in a shaking incubator. 1ml of shaking incubated Agrobacterium suspension was transferred to 50 ml YEP broth containing Km lOOmg/L and incubated for 16 hours as the above condition.
  • the O.D (optical density) of the cultured Agrobacterium suspension was adjusted to 0.8- 1.0 and was used in fransforming the tobacco.
  • Step 5 Construction of stsy cDNA inserted recombinant vector, pBIstsyyl, which is used for transformation.
  • pBIstsyvl To construct a recombinant vector pBIstsyvl, stsy cDNA clone was obtained by RT-PCR and the open reading frame region and some untranslated region was digested by restriction enzyme. Subsequently, it was inserted into the downstream of the CaMV 35S promoter of the pBI 121 vector, which the GUS gene was eliminated(FIG. 2). This recombinant vector was introduced into step4 Agrobacterium producing transformed strain, pBIstsyvl/LBA44040
  • the fransformed Agrobacterium pBIstsyvl/LBA44040 was deposited to the Korean inoculum society on June 18, 2001(KCCM Accession NO.
  • Example 2 Inoculation of transformed Agrobacterium and plant redifferentiation by tissue culture of tobacco
  • Stepl inoculation of fransformed Agrobacterium Grown Nicotiana tabacum L. Wisconsin 38 or Nicotiana tabacum L.
  • Xanthi was selected as a material and the leaf was cross-sectioned. To avoid oxidating and drying, it was stored at a petri-dish that contained 20 ml of broth, made of the MS media added with BA 2.0mg/L, IBA O.Olmg/L, sucrose 30g/L, agar 8g/L and adjusted to pH 5.8. Put 20ml of Agrobacterium suspension and the prepared leaf explant cultured on the YEP in a petri-dish to dip in the suspension fully and shake the petri- dish carefully for 5 minutes at 25 ⁇ 2°C .
  • the explant was placed on the sterilized Whatman No.2 paper and the excessive Agrobacterium was removed. A small amount of explant taken out of the petri-dish not to dry the explant was placed to the antibiotic added callus inducing media. The explant was incubated until the shoot was induced from callus and subcultured every 2 weeks at 25 ⁇ 2°C under continuous lightening. 2 weeks after inoculating on a antibiotic added callus inducing media, callus was starting to form from the fransformed Agrobacterium and the leaf explant. After 3 weeks, callus was generating shoots and getting thicker.
  • Step 2 Rooting the regenerated fransformed shoot.
  • Example 3 genomic PCR, RT-PCR of genomic DNA and total RNA isolated from fransformed tobacco and reverse phase HPLC of resveratrol synthetic substance
  • a genomic DNA was isolated from the transformed novel tobacco leaf and the genomic PCR was performed.
  • Resveratrol was extracted using the Siemann and Cress method. l ⁇ 5g of tobacco leaf was add in a 3ml ethyl acetate test tube and vortexed for 15 seconds. After that the test tube was stored at 4 for several seconds and then at -20, the organic solvent layer was separated with the pasteur pipette while the aqueous solution was extracted in 2ml twice with ethyl acetate to separate the organic solvent layer. Hydrating with anhydrous sodium sulfate, the organic solvent phase was concenfrated using Rotavopor in the low-pressure conditions. The extract was dissolved in methanol and was analyzed with HPLC by the Celotti method.
  • Partisphere C18 5 m(125 X 4.6 X mm I.D., Whatman) was used for a column and water/acetic acid / acetonitrile(75:5:20) for mobile phase. Also the flow rate was adjusted experimentally. For standard, frans-resverafrol(Sigma R5010), catechin(Sigma C1251), epicatechin(Sigma E1753), rutin(Sigma R5143), quercetin(Sigma Q0125) was used.
  • the present invention provides a novel transgenic tobacco produced by introducing the grape resveratrol biosynthesis gene that has strong anti-mutation, anti-oxidation and anti-cancer effects to tobacco. Also genes that have been used for the breed development have the original anti-biotic and anti-virus effects so that this invented product can reduce damages of disease and insect pest in cultivating tobacco and produce mass production of a good quality of tobacco. Further more, this also promotes people's health by alleviating the harmful toxic components of tobacco so that inhibits lung cancer caused by smoking and can be contributed usefully in the seed industry. Applicant's or agenf s International applicationNo. file reference YL01014PCT PCT/K 01/01113

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  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Insects & Arthropods (AREA)
  • Pest Control & Pesticides (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Manufacture Of Tobacco Products (AREA)

Abstract

Cette invention se rapporte à un nouveau tabac transgénique qui restreint l'activité de ses substances nocives en utilisant le gène de biosynthèse de resvératrol Vitis vinifera X Vitis labrusca L. cv. Kyoho.
PCT/KR2001/001113 2000-06-29 2001-06-28 Nouveau tabac transgenique restreignant l'activite de ses substances nocives Ceased WO2002000837A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001269546A AU2001269546A1 (en) 2000-06-29 2001-06-28 Novel transgenic tobacco restraining activity of harmful materials thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20000036450 2000-06-29
KR2000/36450 2000-06-29
KR10-2001-0036939A KR100423072B1 (ko) 2000-06-29 2001-06-27 거봉포도의 레스버라트롤 생합성 유전자를 이용하여 형질전환된 담배 독성 물질의 억제 기능이 있는 신품종 담배
KR2001/36939 2001-06-27

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WO2002000837A2 true WO2002000837A2 (fr) 2002-01-03
WO2002000837A3 WO2002000837A3 (fr) 2002-06-27

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PCT/KR2001/001113 Ceased WO2002000837A2 (fr) 2000-06-29 2001-06-28 Nouveau tabac transgenique restreignant l'activite de ses substances nocives

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4107396A1 (de) * 1990-06-29 1992-01-02 Bayer Ag Stilbensynthase-gene aus weinrebe
CN1033717C (zh) * 1994-03-17 1997-01-01 中国科学院长春应用化学研究所 常温co气体传感器的制造方法

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AU2001269546A1 (en) 2002-01-08

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