WO2008069318A1 - Plante à tolérance au bore produite par fourniture de cette tolérance en excès à une plante, procédé de production de cette plante, et gène destiné à une telle production - Google Patents

Plante à tolérance au bore produite par fourniture de cette tolérance en excès à une plante, procédé de production de cette plante, et gène destiné à une telle production Download PDF

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Publication number
WO2008069318A1
WO2008069318A1 PCT/JP2007/073705 JP2007073705W WO2008069318A1 WO 2008069318 A1 WO2008069318 A1 WO 2008069318A1 JP 2007073705 W JP2007073705 W JP 2007073705W WO 2008069318 A1 WO2008069318 A1 WO 2008069318A1
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Prior art keywords
boron
protein
gene
degraded
endocytosis
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PCT/JP2007/073705
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English (en)
Japanese (ja)
Inventor
Toru Fujiwara
Kyoko Miwa
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Japan Science And Technology Agency
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Priority to JP2008548355A priority Critical patent/JPWO2008069318A1/ja
Publication of WO2008069318A1 publication Critical patent/WO2008069318A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to a BOR4 protein that is not degraded by endocytosis in a boron-excess condition, or a protein selected from the group of 4 or more even sequence numbers that are not degraded by endocytosis in a boron-excess condition.
  • the present invention relates to an excessive boron-tolerant plant into which a DNA (construct) linked with a gene containing a sequence is introduced.
  • the cauliflower mosaic violet-less 35S RNA promoter that induces constitutive expression in plant bodies is an excretory boron transporter protein that is not degraded even under boron-excess conditions, that is, the BOR4 protein or even numbers after 4 in the sequence listing.
  • the present invention relates to a transgenic plant into which a DNA to which a BOR4 gene encoding a protein selected from the described protein group or a gene selected from a gene group having an odd sequence number of 3 or more is linked is introduced.
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2005-253430 discloses that BOR1 can more efficiently control boron uptake from the environment and transport of boron in a living body, and BOR1 and its homologues.
  • a construct in which the cDNA of the gene (BOR2-4) is linked downstream of the GAL1 promoter is constructed, introduced into yeast, and cultured in a medium containing boron at various concentrations for 60 minutes.
  • the yeast that expressed At3 g06450 (BOR3) and Atlgl 5460 (BOR4) was more resistant to boric acid than the vector control, and BOR1 was expressed in the yeast that expressed BOR4. It is stronger than the expressed mother! /, Mentioning that it showed resistance!
  • Patent Document 2 refers to [0028] transformed plants, there is no description suggesting that BOR4 is expressed in transformed Arabidopsis to provide an excess boron-resistant Arabidopsis thaliana.
  • BOR1 we created a transgenic Arabidopsis that overexpressed BOR1 protein linked with green fluorescent protein (GFP) under the control of cauliflower mosaic virus 35S RNA promoter, and observed its behavior according to boron nutrition. Under the condition of boron accumulation in the environment, the accumulation of the cell membrane is observed under boron deficiency conditions. When boron is applied, it is rapidly degraded in about 2 hours, and BOR1-GFP in the degradation process.
  • GFP green fluorescent protein
  • Patent Document 1 JP 2002-262872
  • Non-Patent Document 1 ⁇ & 11 ⁇ 01.420 ⁇ 0.6913 .337-340, 21 November 2002
  • Patent Document 2 JP 2005-253430
  • Non-Patent Document 2 Root Research 13 (2): 51-55 (2004)
  • Non-Patent Document 3 PNAS, Vol.102, No.34, pp.12276-12281,23 August 2005
  • Non-Patent Document 4 The Plant Journal, Vol.46, pp.1084-1091, 2006
  • the problem to be solved by the invention of the present application is that, as described above, boron, which is an essential element for plants, has a narrow optimum concentration range in plants, and the concentration of boron in the soil in which plants are grown. Since accumulation varies depending on geographical conditions and climatic conditions, it is considered that imparting tolerance to excess boron to plants is important for stable production of plants. It is to find a technique for imparting excessive boron tolerance to plants, and to provide a boron excessive tolerance plant. However, until now, there has been no known gene or molecule that imparts excessive boron tolerance to plants, or a production technique using the gene or molecule.
  • the first of the present invention is (1) selected from the group of an even number of sequence numbers that encodes a protein that is not degraded by endocytosis in an excess boron condition and that is not degraded by endocytosis in an excess boron condition.
  • Endothelial cis in a boron-excess condition in which one or several amino acids of the protein are deleted, substituted or added It is a gene sequence that encodes a protein that is not degraded by.
  • the second of the present invention is (2) BOR that is not decomposed by endocytosis in an excess boron condition.
  • the protein that is not degraded by endocytosis under boron-excess conditions is a BOR4 protein
  • the gene is a BOR4 gene that encodes a protein that is not degraded by endocytosis.
  • the plant is an excess boron-tolerant plant, and more preferably (4) a promoter that induces excessive constitutive expression of a gene encoding a protein that is not degraded by endocytosis under boron-excess conditions.
  • the hyperboron-tolerant plant according to (2) or (3) above, which is a mosaic virus 35S RNA promoter), and more preferably, (5) a DNA sequence (construct) is characterized by agrobacterium having TiPlasmid.
  • the agrobacterium used in the present invention is an agrobacterium having a Ti Plasmid. Agrobataterium infects plants and inserts T-DNA on Ti-Plasmid into the chromosomal genome of the plant and recombines the gene. Use the T-DNA to insert the desired construct. By infecting plants with Agrobataterium, we can obtain genetically modified plants with the desired construct.
  • a third aspect of the present invention is (6) BOR which is not decomposed by endocytosis in an excess boron condition.
  • the protein that is not degraded by endocytosis in a boron-excess condition is a BOR4 protein
  • the gene is a BOR4 gene that encodes a protein that is not degraded by the endocytosis.
  • a promoter that induces excessive constitutive expression of a gene encoding a protein that is not degraded by endocytosis in a boron-excess condition is a cauliflower mosaic virus.
  • the method for producing an excessive boron-tolerant plant described in (6) or (7) above which is a 35S RNA promoter (Cadiflower mosaic virus 35S RNA promoter), more preferably, (9) a DNA sequence (constantat) is (6), (7) characterized by being transformed by agrobacterium teratium with TiPlasmid ) Or the method for producing an excessive boron-tolerant plant described in (8).
  • boron transporter BOR4 protein (SEQ ID NO: 2) that is not degraded even under boron-excess conditions in plants is expressed downstream of a promoter that induces constitutive gene expression.
  • BOR4 gene BOR4 gene “Atlgl 5460 gene” (SEQ ID NO: 1) received from RIKEN)
  • AtBOR4 has been found to have an activity to excrete boron with excess boron tolerance in plants.
  • even number sequences are substituted for BOR4 protein.
  • transgenic plant to which the boron excess tolerance is imparted by using a protein having a number and an odd-numbered gene having a sequence number in place of the BOR4 gene.
  • the even-numbered protein and the odd-numbered gene are sequences not present in the BOR1 protein and the BOR1 gene.
  • the transformation step is carried out by introducing a recombinant expression vector into a plant individual so that it is not degraded even under boron-excess conditions! /, And boron transporter protein is expressed. Just do it.
  • the expression vector construction step is performed by constructing a recombinant expression vector comprising a gene encoding a boron transporter protein that is not degraded in a boron-rich environment in a plant, a promoter, and a terminator.
  • a recombinant expression vector comprising a gene encoding a boron transporter protein that is not degraded in a boron-rich environment in a plant, a promoter, and a terminator.
  • various conventionally known vectors as the base vector of the recombinant expression vector.
  • a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant individual or plant cell to be introduced and the introduction method.
  • pBR322, pUC19, pBluescript, pBI vectors and the like can be raised.
  • the method for introducing a vector into a plant is a method using agrobacterium, it is preferable to use a pBI binary vector.
  • pBI binary vectors include pBIN19, pBI121, and pBI221. The power of S
  • the promoter is not particularly limited as long as it is a promoter capable of constitutively expressing a gene in a plant body, particularly inducing strong expression at the root, and a known promoter can be appropriately used.
  • a known promoter can be appropriately used.
  • Specific examples include cauliflower mosaic Winores 35S RNA promoter, actin promoter, nopaline synthase promoter, PRla gene promoter, ribulose 1,5-diphosphate carboxylase 'oxidase small subunit promoter, and the like. it can.
  • the cauliflower monomosaic virus 35S RNA promoter can be more preferably used.
  • the recombinant expression vector may further contain another DNA segment.
  • the other DNA segments are not particularly limited, but may include a terminator, a selection marker, an enhancer, and the like.
  • the terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one.
  • the transcription termination region of the nopaline synthase gene (Nos terminator 1)
  • the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator 1)
  • Nos terminator can be used more preferably.
  • a drug resistance gene can be used as the selection marker.
  • Specific examples include drug resistance genes for hygromycin, kanamycin and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body growing in the medium containing the antibiotic.
  • plasmid In addition to the Ti plasmid (Tumor inducing plasmid), pSPOR Tl, pT7Blue— p vector, pIGl 21-Hm [Plant Cell Report, 15, 809-814 (1995)] And plasmids such as pBI121 [EMBO J. 6, 3901-3907 (1987)], or plant virus vectors such as tobacco mosaic virus, cauliflower mosaic virus, and gemini virus.
  • the transformation step performed in the present invention is a step in which the recombinant expression vector is introduced into a plant individual and is not degraded even under boron-excess conditions! /, And a boron transporter protein is expressed.
  • plant transformation should be carried out using publicly known methods such as the agrobatterium method, leaf disk co-cultivation method, electoral position method, particle gun method, etc. Can do.
  • the ability to list the agrobatterium method as preferred.
  • it is also possible to adopt a method for producing a transformed plant by enhancing the permeability of a plant cell physically or chemically and incorporating the recombinant vector of the present invention directly into a receptor cell.
  • the method for selecting an appropriate transformant after transformation is not particularly limited, and can be based on drug resistance such as hygromycin resistance or a boron-excess medium. It is also possible to select as an index the improvement in growth.
  • the full-length cDNA of Arabidopsis BOR4 was isolated by RIKEN and donated with clone RAF L09-10-J20. PCR was performed to amplify an ORF (open reading frame) region that does not contain a 5 'UTR region upstream and does not contain a stop codon downstream.
  • the primer used included a Kpnl site on the upstream side and an Ncol site on the downstream side, and GGA GGA GGA GGS GCC (Gly Gly Gly Gly Ala) and a linker region were added to the portion corresponding to the stop codon.
  • the amplified ORF region of BOR4 was cleaved with the corresponding restriction enzyme, and the upstream gene was linked to the force-refractor mosaic virus 35S promoter (35S), and the downstream gene encoding GFP and Nos terminator (tNOS).
  • 35S-BOR4-GFP-tNOS DNA was amplified 5 'with a primer having a CACC sequence and incorporated into pENTR-TOPO of Invitrogen (USA). This is recombined into Gateway vector pMDC99 (Curtis, MD, Grossniklaus, U. (2003). A g ateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462-469.) The vector was used. PMDC99 has a hygromycin resistance gene in the T-DNA region.
  • agrobataterum into which an expression vector has been introduced is added to a 2001111 medium containing antibiotics (kanamycin SO ⁇ ug / ml, rifampicin 5 8/1111): 6 & 0— 6 pton 10 g, Bacto yeast extract 10 g, NaCl 5 g / lL pH was cultured in 7 to 7.2). Subsequently, the cells were collected from the culture and suspended in 400 ml of an infectious medium (5% sucrose, a little MgCl, 0.05% Tween20).
  • antibiotics kanamycin SO ⁇ ug / ml, rifampicin 5 8/1111
  • Next-generation seeds ( ⁇ 2) were collected from the above hygromycin-resistant plant (T1) and sown in the same hygromycin selection medium, and hygromycin-resistant: a line with a sensitivity of 3: 1 was selected. did. Next-generation seeds ( ⁇ 3) were collected from plant individuals that showed resistance in this line ( ⁇ 2), sown in hygromycin selection medium, and strains in which all individuals became hygromycin-resistant were copied by DNA. Yes, it was obtained as a strain with homologous DNA insertion. [0030] (3) BOR4 protein accumulation in transformants
  • Three lines (line numbers 1, 4, and 5) of T3 seeds of transformants having the T DNA obtained above in homozygous were sown in a solid medium containing 100 M boric acid and cultivated for 24 days.
  • the solid medium is a MGRL (Fujiwara et al., Plant Physiol. 99, 263-268 ⁇ 1992) solution with 2% sucrose and 0.2% gellan gum. Thereafter, the plant individuals were transplanted into MG RL solution (without sucrose and gellan gum) and grown for 6 days.
  • the boron excess tolerance test was performed on the MGRL solid medium.
  • a solid medium containing 2% sucrose, 1.5% gellan gum, 0.03, 3, 6, 10 mM boric acid in a MGRL (Fujiwa ra et al., Plant Physiol. 99, 263-268.1992) solution was prepared.
  • Surface-sterilized transformant seeds (35S-BOR4-GFP transgenic) and wild-type seeds (Co 0) were sown, and growth was observed after 17 days.
  • Degradation of the BOR4 protein under boron-excess conditions involves amino acids from the 6th to the 11th amino acid sequence shown in SEQ ID NO: 20 (the 407th force of the protein of SEQ ID NO: 2 also corresponds to the 412th amino acid).
  • the results show that boric acid resistance can be imparted by proteins other than the B0R4 protein that has this sequence and a sequence that has the same effect as this sequence. .
  • a gene linked to a DNA encoding a protein such as the excretory boron transporter protein BOR4 that is not degraded even under boron-excess conditions is included downstream of a promoter that induces constitutive gene expression in plants.
  • Transgenic plants with improved resistance to excess boron are provided.
  • a transgenic plant having improved resistance to excess boron, comprising DNA in which a gene encoding a protein such as BOR4 is linked downstream of a promoter that induces constitutive expression.

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Abstract

Plante à tolérance au bore en excès, procédé de production de cette plante, et gène destiné à une telle production. Ladite plante comporte une séquence d'ADN (construction) introduite, laquelle comprend: promoteur capable d'induire l'expression constitutive en excès de protéine BOR4 ou de protéine appartenant aux protéines décrites dans les numéros d'identification de séquence ayant des numéros pairs égaux ou supérieurs à 4; et gène BOR4 ou gène appartenant aux gènes décrits dans les numéros d'identification de séquence ayant des chiffres impairs égaux ou supérieurs à 3 et à ligature en aval avec le promoteur.
PCT/JP2007/073705 2006-12-07 2007-12-07 Plante à tolérance au bore produite par fourniture de cette tolérance en excès à une plante, procédé de production de cette plante, et gène destiné à une telle production WO2008069318A1 (fr)

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JP2008548355A JPWO2008069318A1 (ja) 2006-12-07 2007-12-07 植物に過剰ホウ素耐性を付与したホウ素耐性植物、前記植物の作出方法及び前記作出に利用する遺伝子

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JP2006330816 2006-12-07
JP2006-330816 2006-12-07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802156A (zh) * 2024-02-29 2024-04-02 云南师范大学 硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002262872A (ja) * 2001-03-06 2002-09-17 Toru Fujiwara ホウ素輸送に関与する遺伝子
JP2005253430A (ja) * 2004-03-15 2005-09-22 Japan Science & Technology Agency ホウ素トランスポーター及びその遺伝子

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002262872A (ja) * 2001-03-06 2002-09-17 Toru Fujiwara ホウ素輸送に関与する遺伝子
JP2005253430A (ja) * 2004-03-15 2005-09-22 Japan Science & Technology Agency ホウ素トランスポーター及びその遺伝子

Non-Patent Citations (11)

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DATABASE GENBANK [online] 19 June 1999 (1999-06-19), Database accession no. (AC007591) *
DATABASE GENBANK [online] 26 December 2001 (2001-12-26), Database accession no. (AY069887) *
HANAOKA H. AND FUJIWARA H.: "Shokubutsu no Boron Yuso Kiko no Shomei to Sono Riyo", BIO INDUSTRY, vol. 23, no. 4, April 2006 (2006-04-01), pages 63 - 68 *
HAYES J.E. AND REID R.J.: "Boron tolerance in barley is mediated by efflux of boron from the roots", PLANT PHYSIOL., vol. 136, 2004, pages 3376 - 3382 *
MIWA K. ET AL.: "Improvement of seed yields under boron-limiting conditions through overexpression of BOR1, a boron transporter for xylem loading, in Arabidopsis", PLANT J., vol. 46, June 2006 (2006-06-01), pages 1084 - 1091 *
MIWA K. ET AL.: "Plants tolerant of high boron levels", SCIENCE, vol. 318, 30 November 2007 (2007-11-30), pages 1417 *
MIWA K. ET AL.: "Thale Cress Boron Transporter BOR4 o Mochiita Boron Kajo Taisei Shokubutsu no sakushutsu", DAI 48 KAI THE JAPANESE SOCIETY OF PLANT PHYSIOLOGISTS NENKAI YOSHISHU, 15 March 2007 (2007-03-15), pages 163 + ABSTR. NO. (2AE04(289)) *
REID R.: "Identification of boron transporter genes likely to be responsible for tolerance to boron toxicity in wheat and barley", PLANT CELL PHYSIOL., vol. 48, 14 November 2007 (2007-11-14), pages 1673 - 1678, XP055235268, DOI: doi:10.1093/pcp/pcm159 *
SUTTON T. ET AL.: "Boron-toxicity tolerance in barley arising from efflux transporter amplification", SCIENCE, vol. 318, 30 November 2007 (2007-11-30), pages 1446 - 1449 *
TAKANO J. ET AL.: "Arabidopsis boron transporter for xylem loading", NATURE, vol. 420, 2002, pages 337 - 340, XP002993426, DOI: doi:10.1038/nature01139 *
TAKANO J. ET AL.: "Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability", PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 12276 - 12281 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802156A (zh) * 2024-02-29 2024-04-02 云南师范大学 硼酸转运蛋白基因在转基因植株筛选中的应用及筛选方法

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