WO2010035784A1 - 植物のバイオマス量を増産させる遺伝子及びその利用方法 - Google Patents
植物のバイオマス量を増産させる遺伝子及びその利用方法 Download PDFInfo
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- 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
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- 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
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- 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
- C12N15/8273—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 drought, cold, salt resistance
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- 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
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- 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 includes a plant into which a predetermined gene has been introduced or a modified expression control region of the endogenous gene, and a biomass amount by introducing the predetermined gene or modifying the endogenous gene expression control region
- the present invention relates to a method for increasing the production of a plant and imparting salt stress tolerance, and a method for producing a plant body in which the amount of biomass is increased and salt stress tolerance is imparted.
- Biomass generally refers to the total amount of organisms that inhabit or exist per certain area, and in particular when plants are targeted, it means the dry weight per unit area.
- the unit of biomass is quantified by mass or energy amount.
- biomass and “biomass” are synonymous with the expression “biomass”.
- Standing crop is sometimes used. Since plant biomass is generated by fixing carbon dioxide in the atmosphere using solar energy, it can be captured as so-called carbon neutral energy. Therefore, increasing plant biomass has the effects of global environmental conservation, prevention of global warming, and reduction of greenhouse gas emissions. Therefore, the technology for increasing plant biomass production is highly industrially important.
- plants are cultivated for the purpose of part of the tissue itself (seed, root, leaf stem, etc.) or for the purpose of producing various substances such as fats and oils.
- fats and oils produced by plants soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn oil, bean flower oil, palm oil, rapeseed oil, etc. have been known since ancient times. Widely used in industrial applications.
- oils and fats produced by plants are also used as raw materials for biodiesel fuel and bioplastics, and their applicability is expanding as petroleum alternative energy.
- a gene having a novel function for greatly improving the amount of plant biomass can be searched for, and a technology for greatly increasing the amount of plant biomass and providing salt stress resistance to the plant body can be provided.
- the purpose is to do.
- a protein having a coiled-coil structure, a nucleic acid binding site, and a leucine-rich repeat structure in the molecule which encodes a protein having a characteristic common sequence
- the present inventors have completed the present invention by finding new knowledge that the amount of plant biomass can be greatly improved and salt stress tolerance can be imparted to a plant body by introducing a gene to be altered or by altering the expression control region of the gene concerned. It came to.
- the plant according to the present invention comprises a coiled-coil structure and a nucleic acid binding site having a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 in this order from the N-terminal side.
- a gene encoding a protein having a leucine rich repeat structure is introduced, or the expression control region of the gene is modified.
- the method for increasing the biomass amount according to the present invention and imparting salt stress tolerance to a plant body includes the N-terminal consensus sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2. This is a method of introducing a gene encoding a protein having a coiled coil structure, a nucleic acid binding site, and a leucine rich repeat structure in this order from the side, or altering the expression control region of the gene concerned.
- the method for producing a plant according to the present invention comprises a coiled-coil structure and a nucleic acid having a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 in this order from the N-terminal side.
- the protein preferably further has a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 1 on the N-terminal side of the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3.
- the gene is AT1G59124, AT1G58807, AT1G59218, AT1G58848, AT1G58602, AT1G58410, AT1G58400, AT1G58390, AT1G59620, AT1G59780, AT1G50180, AT1G53350, AT5G43470, AT5G10920, AT1G5820 Alternatively, it can be a gene functionally equivalent to the gene.
- the gene preferably encodes one of the following proteins (a) to (c).
- the functionally equivalent gene is a gene encoding a coiled-coil structure derived from an organism other than Arabidopsis thaliana, a nucleic acid binding site, and a protein having a leucine-rich repeat structure. Raise be able to. Examples of organisms other than Arabidopsis thaliana, a nucleic acid binding site, and a protein having a leucine-rich repeat structure. Raise be able to. Examples of organisms other than Arabidopsis thaliana, a nucleic acid binding site, and a protein having a leucine-rich repeat structure. Raise be able to. Examples of organisms other than Arabidopsis thaliana, a nucleic acid binding site, and a protein having a leucine-rich repeat structure. Raise be able to. Examples of organisms other than Arabidopsis thaliana, a nucleic acid binding site, and a protein having a leucine-rich repeat structure. Raise be able to. Examples of
- Examples of plants targeted in the present invention include dicotyledonous plants such as Arabidopsis plants and Brassicaceae plants.
- the target plant in the present invention includes monocotyledonous plants such as rice and sugar cane among grasses and gramineous plants.
- the plant according to the present invention has a significantly increased biomass amount compared to the wild type, and has been given resistance to salt stress. Further, the method for increasing the biomass amount according to the present invention and imparting salt stress tolerance to the plant body can greatly increase biomass production as compared with the wild type of the target plant, and also has salt stress tolerance. Can be granted. Further, the method for producing a plant according to the present invention can produce a plant having a significantly improved biomass amount and resistance to salt stress compared to the wild type. Therefore, by applying the present invention, for example, it is possible to achieve a reduction in cost by achieving an improvement in productivity when the plant itself is used as a product, and also in a high salt concentration environment. Can be nurtured.
- the plant according to the present invention introduces a gene encoding a protein having a coiled-coil structure having a characteristic common sequence, a nucleic acid binding site, and a leucine-rich repeat structure (hereinafter abbreviated as CC-NBS-LRR) into the plant. Or a modified expression control region of the gene of interest, and the amount of biomass is significantly improved as compared to the wild type, and salt stress tolerance is conferred. Significantly increase the expression level of the target gene compared to the wild-type expression level by introducing the target gene exogenously into the plant or modifying the expression control region of the endogenous gene be able to.
- CC-NBS-LRR leucine-rich repeat structure
- the plant according to the present invention may be one in which the CC-NBS-LRR gene is expressed throughout the plant tissue, or may be expressed in at least a part of the plant tissue. good.
- the plant tissue is meant to include plant organs such as leaves, stems, seeds, roots and flowers.
- the expression control region means a promoter region to which RNA polymerase binds and a region to which other transcription factors bind.
- the transcription control region it is preferable to replace, for example, the promoter region in the endogenous transcription control region with a promoter region capable of higher expression.
- the CC-NBS-LRR gene has a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 in this order from the N-terminal side. Encodes the CC-NBS-LRR protein.
- CC-NBS-LRR has a nucleic acid binding site as described in Reference (1) (The Plant Cell, Vol. 15, 809-834, April 2003). (Also called R protein).
- CC-NBS-LRR having the above-mentioned common sequence is classified as “CNL-D” in the above-mentioned reference, and is different in that it has a coiled-coil structure, a nucleic acid binding site and a leucine-rich repeat structure from the N-terminal side.
- CNL-D CC-NBS-LRR
- part of the nucleic acid binding motif between the coiled-coil structure and the leucine-rich repeat structure is characteristic, and has a biological function different from other CC-NBS-LRRs. It is thought to have.
- CC-NBS-LRR classified as CNL-D has regions (3) and (2) that are highly conserved in order from the N-terminal side. That is, these highly conserved regions (3) and (2) can be defined as a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2.
- the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 are sequences characteristic of CNL-D in the above-mentioned classification of CC-NBS-LRR, It is an arrangement that serves as a clear distinction from other groups.
- CC-NBS-LRR classified as CNL-D was highly conserved on the N-terminal side of region (3) in addition to regions (3) and (2), as shown in FIG. It can be seen that it has region (1). That is, this highly conserved region (1) can be defined as a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 1. That is, CC-NBS-LRR classified as CNL-D includes the amino acid sequence shown in SEQ ID NO: 3 in addition to the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 and the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2. It can also be defined as having a common sequence consisting of the amino acid sequence shown in SEQ ID NO: 1 on the N-terminal side of the common sequence consisting of sequences.
- the amino acid residue represented as Xaa is an arbitrary amino acid and is not limited to any amino acid.
- the third amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is isoleucine (three letter notation: Ile, one letter notation: I, the same shall apply hereinafter), leucine (Leu, L) or valine (Val, V) is preferred.
- the fourth amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably threonine (Thr, T), serine (Ser, S), or alanine (Ala, A).
- the sixth amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably methionine (Met, M) or leucine (Leu, L).
- the ninth amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably leucine (Leu, L) or isoleucine (Ile, I).
- the 16th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably lysine (Lys, K) or arginine (Arg, R).
- the 18th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably valine (Val, V) or isoleucine (Ile, I).
- the 20th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably asparagine (Asn, N), aspartic acid (Asp, D) or histidine (His, H).
- the 22nd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably glutamic acid (Glu, E) or aspartic acid (Asp, D).
- the amino acid residue represented as Xaa is an arbitrary amino acid and is not limited to any amino acid.
- the second amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably histidine (His, H), arginine (Arg, R), or glutamine (Gln, Q).
- the third amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably leucine (Leu, L) or methionine (Met, M).
- the sixth amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably methionine (Met, M), isoleucine (Ile, I) or leucine (Leu, L).
- the seventh amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably methionine (Met, M) or valine (Val, V).
- the 10th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably valine (Val, V) or isoleucine (Ile, I).
- the 12th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably leucine (Leu, L) or isoleucine (Ile, I). That is, the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 is more specifically C (H / R / Q) (L / M) HD (M / I / L) (M / V) RE (V / I) C (L / I) is preferred.
- a plurality of amino acids in parentheses indicate possible amino acid residue variations at that position.
- the amino acid residue represented as Xaa is an arbitrary amino acid and is not limited to any amino acid.
- the second amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine (Ile, I) or leucine (Leu, L).
- the sixth amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V) or alanine (Ala, A).
- the seventh amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably arginine (Arg, R) or lysine (Lys, K).
- the 10th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably methionine (Met, M) or leucine (Leu, L).
- the 11th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V) or isoleucine (Ile, I). That is, the common sequence consisting of the amino acid sequence shown in SEQ ID NO: 3 is more specifically Y (I / L) EEL (V / A) (R / K) RN (M / L) (V / I). It is preferable. In this amino acid sequence, a plurality of amino acids in parentheses indicate possible amino acid residue variations at that position.
- reference (3) Henikoff S., Henikoff JG, Amino-acid substitution matrices from protein blocks, Proc.Natl. Acad.Sci. USA, 89, 10915-109191992 (1992), Fig. A score matrix (BLOSUM) for substitution mutations of amino acid residues was proposed and widely used in .2.
- amino acid substitution between similar side chain chemical properties is based on the knowledge that structural and functional changes given to the whole protein are reduced.
- the side chain groups of amino acids considered in multiple alignment can be considered based on indicators such as chemical properties and physical size.
- score matrix (BLOSUM) disclosed in reference (3) as an amino acid group having a score of 0 or more, preferably a group of amino acids having a value of 1 or more.
- the following eight groups are listed as typical groups.
- Other fine groupings may be any amino acid group of 0 or more, preferably 1 or more, more preferably 2 or more amino acid groups of the score value.
- Aliphatic hydrophobic amino acid group This group is a group of amino acids having a hydrophobic hydrophobic side chain among the neutral non-polar amino acids shown in the above-mentioned reference (1), V (Val, valine), L (Leu, leucine) , I (Ile, isoleucine) and M (Met, methionine).
- V Val, valine
- L Leu, leucine
- I Ile, isoleucine
- M Metal, methionine
- FGACWP is not included in this “aliphatic hydrophobic amino acid group” for the following reasons. This is because G (Gly, glycine) and A (Ala, alanine) are less than a methyl group and have a weak nonpolar effect.
- C Cys, cysteine
- F Phenylalanine
- W Trp, tryptophan
- P Pro, proline
- ST group Group with hydroxymethylene group
- S Ser, serine
- T Thr, threonine
- Acidic amino acids This group is a group of amino acids having an acidic carboxyl group in the side chain, and is composed of D (Asp, aspartic acid) and E (Glu, glutamic acid).
- KR group This group is a group of basic amino acids and is composed of K (Lys, lysine) and R (Arg, arginine). These K and R are positively charged and have basic properties over a wide pH range. On the other hand, H (His, histidine) classified as a basic amino acid is not classified into this group because it is hardly ionized at pH 7.
- Methylene group polar group (DHN group) This group has a feature that a methylene group is bonded as a side chain to a carbon element at the ⁇ -position and has a polar group at the tip.
- Dimethylene group polar group (EKQR group) This group has a feature that a linear hydrocarbon having a dimethylene group or more is bonded as a side chain to a carbon element at the ⁇ -position and has a polar group at the tip.
- E Glu, glutamic acid, polar group is carboxyl group
- K Lis, lysine, polar group is amino group
- Q Gln, glutamine, polar group is amide group
- R Arg, arginine, polar group is imino group
- Aromatic (FYW Group) This group is an aromatic amino acid with a benzene nucleus in the side chain and is characterized by aromatic chemical properties. It consists of F (Phe, phenylalanine), Y (Tyr, tyrosine), W (Trp, tryptophan).
- Circular & polar (HY group) This group is an amino acid that has a cyclic structure in the side chain and also a polarity, H (H, histidine, both cyclic structure and polar group are imidazole groups), Y (Tyr, tyrosine, cyclic structure is polar with benzene nucleus The group consists of hydroxyl).
- the amino acid residue shown as Xaa may be any amino acid, but the amino acid residue shown as Xaa is within the group of 1) to 8) above. It can be seen that amino acid substitution may be performed. That is, in the present invention, as long as the CC-NBS-LRR gene has two common sequences consisting of the amino acid sequences shown in SEQ ID NOs: 3 and 2, preferably three common sequences consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3. Any plant-derived CC-NBS-LRR gene may be used.
- the CC-NBS-LRR gene having a common sequence consisting of the predetermined amino acid sequences shown in SEQ ID NOs: 1 to 3 in Arabidopsis thaliana includes AT1G59124, AT1G58807, AT1G59218, AT1G58848, AT1G58602, AT1G58410, AT1G58400, AT1G58390, AT1G59620 , AT1G59780, AT1G50180, AT1G53350, AT5G43470, AT5G48620, AT5G35450 and AT1G10920.
- at least one gene selected from these gene groups is introduced into the plant body, or the expression control region of the endogenous gene is modified. To do.
- the gene identified by AT1G59124, AT1G58807, AT1G59218, AT1G58848 and AT1G58602 is introduced into the plant body, or the expression control region of the endogenous gene is modified. To do.
- the base sequence of the coding region in the gene specified by AT1G58602 is shown in SEQ ID NO: 4, and the amino acid sequence of CC-NBS-LRR encoded by the gene specified by AT1G58602 is shown in SEQ ID NO: 5.
- genes functionally equivalent to the genes listed above may be introduced into the plant body.
- the functionally equivalent gene includes, for example, a gene encoding CC-NBS-LRR which is derived from an organism other than Arabidopsis thaliana and has two common sequences consisting of the amino acid sequences shown in SEQ ID NOs: 3 and 2. It is.
- a functionally equivalent gene is a protein having CC-NBS-LRR and encoding an R protein that interacts directly or indirectly with an effector.
- Examples of organisms other than the above Arabidopsis include grapes (Vitis vinifera). More specifically, the gene identified by accession number A7Q3G8, the gene identified by A5BY93, the gene identified by A7Q3G6, the gene identified by A5C0R9, and the gene identified by A7Q3H1 as the genes of Vitis vinifera Can be mentioned.
- a gene encoding CC-NBS-LRR having two common sequences consisting of the amino acid sequences shown in SEQ ID NOs: 3 and 2 in plants other than Arabidopsis thaliana represented by these is encoded by the AT1G58602 gene derived from Arabidopsis thaliana listed above. Based on the amino acid sequence, it can be easily searched and identified from a known database such as GenBank.
- the CC-NBS-LRR gene is not limited to the CC-NBS-LRR gene consisting of the base sequence and amino acid sequence specified by the sequence numbers as described above. That is, the CC-NBS-LRR gene includes an amino acid sequence in which one or a plurality of amino acid sequences are deleted, substituted, added or inserted in the amino acid sequence specified by the sequence number as described above, and CC It may have an activity that functions as -NBS-LRR.
- the plurality of amino acids for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, particularly preferably 1 to 3 are used. means.
- amino acid deletion, substitution, or addition can be performed by modifying the base sequence encoding the CC-NBS-LRR gene by a technique known in the art.
- Mutation can be introduced into a nucleotide sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant- Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA Bio Inc.) or the like, or using LA PCR-in-vitro Mutagenesis series kits (trade name, manufactured by TAKARA Bio Inc.).
- EMS ethyl methanesulfonic acid
- 5-bromouracil 2-aminopurine
- hydroxylamine N-methyl-N'-nitro-N nitrosoguanidine
- other carcinogenic compounds are representative.
- a method using a chemical mutagen such as that described above may be used, or a method using radiation treatment or ultraviolet treatment represented by X-rays, alpha rays, beta rays, gamma rays and ion beams may be used.
- the CC-NBS-LRR gene may be a homologous gene of the CC-NBS-LRR gene consisting of the base sequence and amino acid sequence specified by the sequence numbers as described above.
- the homologous gene generally means a gene that has evolved and branched from a common ancestral gene, and is generated by two kinds of homologous genes (ortholog) and duplicated branching within the same species. It means to include homologous genes (paralog).
- the above-mentioned “functionally equivalent gene” includes homologous genes such as orthologs and paralogs.
- the above-mentioned “functionally equivalent genes” include genes that do not evolve from a common gene but simply have similar functions.
- the similarity to these amino acid sequences is, for example, 70% or more, preferably 80% or more
- the similarity value means a value obtained by default setting using a computer program in which a BLAST (Basic Local Alignment Search Tool) program is implemented and a database storing gene sequence information.
- a gene similar to the CC-NBS-LRR2C gene consisting of the base sequence and amino acid sequence specified by the sequence number as described above the genome from the target plant if the plant genome information is not clear
- constructing a cDNA library of the target plant and stringent to at least a part of the CC-NBS-LRR gene consisting of the base sequence and amino acid sequence specified by SEQ ID NO as described above It can be identified by isolating a genomic region or cDNA that hybridizes under various conditions.
- stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, hybridization at 45 ° C.
- Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. OleMolecular lonCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
- the plant according to the present invention introduces a gene encoding CC-NBS-LRR having two common sequences consisting of the amino acid sequences shown in SEQ ID NOs: 3 and 2 into the plant body, or an expression control region of the gene concerned. By altering, the amount of biomass is significantly improved as compared to the wild type, and the salt stress tolerance is obtained.
- a method for introducing the CC-NBS-LRR gene into a plant a method for introducing an expression vector in which an exogenous CC-NBS-LRR gene is placed under the control of a promoter capable of expression in the plant can be mentioned. be able to.
- Examples of a technique for modifying the expression control region of the gene of interest include a technique for modifying the promoter of the endogenous CC-NBS-LRR gene in the target plant.
- a method of introducing an expression vector in which the CC-NBS-LRR gene described above is arranged under the control of a promoter that enables expression in a plant body is preferable.
- the expression vector expression vector is constructed so as to include a promoter that enables expression in a plant body and the CC-NBS-LRR gene described above.
- Various vectors known in the art can be used as the base vector for the expression vector.
- a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method.
- Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI vectors.
- the method for introducing a vector into a plant is a method using Agrobacterium, it is preferable to use a pBI binary vector.
- Specific examples of the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.
- the promoter is not particularly limited as long as it is a promoter capable of expressing the CC-NBS-LRR gene in a plant body, and a known promoter can be suitably used.
- promoters include cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase Oxidase small subunit gene promoter, napin gene promoter, etc.
- cauliflower mosaic virus 35S promoter, actin gene promoter, or ubiquitin gene promoter can be more preferably used. When each of the above promoters is used, any gene can be strongly expressed when introduced into a plant cell.
- a promoter having a function of expressing in a site-specific manner in a plant can be used.
- any conventionally known promoter can be used.
- the expression vector may further contain other DNA segments in addition to the promoter and the CC-NBS-LRR gene.
- the other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency.
- the recombinant expression vector may further have a T-DNA region.
- the T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the recombinant expression vector into a plant body.
- the transcription 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 (Nos terminator) of the nopaline synthase gene the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used. Of these, the Nos terminator can be more preferably used.
- the transcription terminator is placed at an appropriate position, and after being introduced into a plant cell, an unnecessarily long transcript is synthesized, and a strong promoter reduces the copy number of the plasmid. Can be prevented.
- a transformant selection marker for example, a drug resistance gene can be used.
- drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like.
- Examples of the base sequence for improving the translation efficiency include an omega sequence derived from tobacco mosaic virus. By placing this omega sequence in the untranslated region (5′UTR) of the promoter, the translation efficiency of the fusion gene can be increased.
- the recombinant expression vector can contain various DNA segments depending on the purpose.
- the method for constructing the recombinant expression vector is not particularly limited, and the promoter, the CC-NBS-LRR gene, the transcription repressor conversion polynucleotide, and, if necessary, the mother vector appropriately selected. What is necessary is just to introduce
- an expression cassette may be constructed by linking the CC-NBS-LRR gene and a promoter (such as a transcription terminator if necessary) and introduced into a vector.
- the order of the DNA segments can be defined by setting the cleavage sites of each DNA segment as complementary protruding ends and reacting with a ligation enzyme.
- the promoter, the CC-NBS-LRR gene, and the terminator may be in order from the upstream.
- the types of reagents for constructing the expression vector that is, the types of restriction enzymes and ligation enzymes are not particularly limited, and commercially available ones may be appropriately selected and used.
- the method for producing the above expression vector is not particularly limited, and a conventionally known method can be used.
- Escherichia coli may be used as a host and propagated in the E. coli.
- a preferred E. coli type may be selected according to the type of vector.
- the expression vector described above is introduced into a target plant by a general transformation method.
- the method (transformation method) for introducing the expression vector into the plant cell is not particularly limited, and any conventionally known method suitable for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of directly introducing into plant cells can be used.
- a method using Agrobacterium for example, Bechtold, E., Ellis, J. and Pelletier, G. (1993) In Planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis plants. CR Acad. Sci. Paris Sci. Vie, 316, 1194-1199.
- a method for directly introducing an expression vector into a plant cell for example, a microinjection method, an electroporation method (electroporation method), a polyethylene glycol method, a particle gun method, a protoplast fusion method, a calcium phosphate method, or the like can be used.
- a transcription unit necessary for expression of the target gene such as a promoter or transcription terminator, and DNA containing the target gene are sufficient, and the vector function Is not mandatory.
- the DNA contains only the protein coding region of the gene of interest that does not have a transcription unit, it is sufficient if it can be integrated without the transcription unit of the host and the gene of interest can be expressed.
- Examples of the plant cell into which the expression vector or the expression cassette containing the gene of interest without the expression vector is introduced include, for example, cells of each tissue in plant organs such as flowers, leaves, roots, callus, suspension culture, etc. A cell etc. can be mentioned.
- the expression vector may be appropriately constructed according to the type of plant to be produced, but a general-purpose expression vector is constructed in advance and introduced into plant cells. Also good.
- the plant to which the expression vector is introduced in other words, the plant to be increased in biomass production is not particularly limited. That is, by introducing the CC-NBS-LRR gene described above, it is possible to expect an effect of increasing biomass production for all plant bodies.
- Examples of the target plant include dicotyledonous plants and monocotyledonous plants, for example, plants belonging to the family Brassicaceae, Gramineae, Eggplant, Legume, Willow, etc. (see below), but are limited to these plants. Is not to be done.
- Brassicaceae Arabidopsis thaliana, Brassica (Brassica rapa, Brassica napus), Cabbage (Brassica oleracea var. Capitata), Rapeseed (Brassica rapa, Brassica napus), Nanohana (Brassica rapa, Brassica ⁇ napus) Pekinensis), Chingen rhinoceros (Brassica rapa var. (Brassica rapa var. Chinensis), Japanese radish (Brassica Raphanus sativus), Wasabi (Wasabia japonica), etc.
- Eggplant family tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato (Lycopersicon lycopersicum), capsicum (Capsicum annuum), petunia (Petunia), etc.
- Legumes soybean (Glycinelymax), pea (Pisum sativum), broad bean (Vicia faba), wisteria (Wisteria floribunda), peanut (Arachis. Hypogaea), Lotus corniculatus var. Japonicus, common bean (gargo ul) (Vigna angularis), Acacia.
- Asteraceae chrysanthemum (Chrysanthemum morifolium), sunflower (Helianthus annuus), etc.
- Palms oil palm (Elaeis guineensis, Elaeis oleifera), coconut (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia) Ursiaceae: Rhis succedanea, Cashew nutcrest (Anacardium occidentale), Urushi (Toxicodendron vernicifluum), Mango (Mangifera indica), Pistachio (Pistacia vera) Cucurbitaceae: pumpkins (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumbers (Cucumis sativus), callas (Trichosanthes cucumeroides), gourds (Lagenaria siceraria var. Gourda) Rosaceae: Almond (Amygdalus communis), Rose (Rosa), Strawberry (Fragaria), Sakura (Prunus), Apple (Malus pumila var. Domestica
- Dianthus Carnation (Dianthus caryophyllus).
- Lily family Tulip (Tulipa), Lily (Lilium), etc.
- biofuels such as sugarcane, corn, rapeseed, and sunflower.
- biofuels such as sugarcane, corn, rapeseed, and sunflower.
- biofuels such as bioethanol, biodiesel, biomethanol, bio DME, bio GTL (BTL) and biobutanol by increasing the biomass of energy crops.
- the CC-NBS-LRR gene that can be used in the present invention can be isolated from various plants and used, but is appropriately selected depending on the type of plant for which biomass production is to be increased. Can be used. That is, when the plant whose biomass production is to be increased is a monocotyledonous plant, it is preferable to introduce a CC-NBS-LRR gene isolated from a monocotyledonous plant.
- a CC-NBS-LRR gene derived from a dicotyledonous plant may be introduced even if the plant whose biomass production is to be increased is a monocotyledonous plant. That is, for example, the Arabidopsis thaliana-derived CC-NBS-LRR gene (SEQ ID NO: 4) may be introduced not only in dicotyledonous plants but also in plants widely classified as monocotyledonous plants.
- a selection step for selecting an appropriate transformant from among plant bodies can be performed by a conventionally known method.
- the selection method is not particularly limited.
- the selection may be performed on the basis of drug resistance such as hygromycin resistance, and after growing the transformant, the plant itself, or any organ or tissue You may select the thing which measured weight and has increased significantly compared with the wild type.
- progeny plants can be obtained from the transformed plants obtained by the transformation treatment according to a conventional method.
- a progeny plant that retains the trait into which the CC-NBS-LRR gene has been introduced or the endogenous expression control region of the CC-NBS-LRR gene has been modified, based on the biomass amount.
- plant cells, seeds, fruits, strains, callus, tubers, cuttings, clumps, and other propagation materials are obtained from the transformed plants and their progeny, and the amount of biomass is increased by having the above traits based on these. It is also possible to mass-produce stable plant lines.
- the plant body in the present invention includes at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed. That is, in this invention, if it is a state which can be made to grow finally to a plant individual, all will be considered as a plant body.
- the plant cells include various forms of plant cells. Such plant cells include, for example, suspension culture cells, protoplasts, leaf sections and the like. Plants can be obtained by growing and differentiating these plant cells. In addition, the reproduction
- the CC-NBS-LRR gene having the above-described specific common sequence is introduced into a plant body, or the expression control region of the CC-NBS-LRR gene existing therein is modified.
- the significant increase in the biomass amount means that the total weight per individual is statistically significantly larger than that of the wild type. At this time, even if some tissues of the plant body were specifically large and other tissues were equivalent to the wild type, if the total weight of the whole plant body was large, it was judged that the biomass amount increased. To do.
- salt stress tolerance means that the upper limit of the salt concentration which can be grown becomes significantly large compared with a wild type.
- salt stress tolerance means that even if the concentration of salt contained in the growth environment such as soil or medium is so high that it is poorly developed or killed in wild type plants, it does not cause poor growth or death.
- the concentration of salt contained in the growth environment such as soil or medium is so high that it is poorly developed or killed in wild type plants, it does not cause poor growth or death.
- the concentration of salt contained in the growth environment such as soil or medium
- the expression control region of the CC-NBS-LRR gene is modified is 300 mM, preferably 250 mM. More preferably, it exhibits salt stress tolerance such that it can grow even in a medium having a salt concentration of 200 mM, most preferably 150 mM.
- the biomass amount of the plant body is increased, either the case where the whole plant body is intended for production or the case where a part of the plant body (for example, seeds) or its contained component is intended for production.
- productivity can be improved.
- the fats and oils contained in plant seeds are intended for production, the amount of fats and oils that can be recovered per planted area can be greatly improved.
- the fats and oils are not particularly limited, and examples thereof include plant-derived fats and oils such as soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn oil, benflower oil, and rapeseed oil. .
- the manufactured fats and oils can be widely used for household use and industrial use, and can also be used as a raw material for biodiesel fuel. That is, according to the present invention, by using the plant body in which the CC-NBS-LRR gene is introduced or the expression control region of the CC-NBS-LRR gene is modified, Industrial fats and oils, biodiesel fuel, etc. can be manufactured at low cost.
- the tolerance of salt stress of the plant body is improved, it is possible to grow in a high salt concentration soil that could not be grown if it was a wild type plant body.
- the high salt concentration soil include coastal soil.
- oil and fat contained in plant seeds are intended for production, high production using high-salt concentration soil, and further, the above-described oil and fat for use in home or industrial use, biodiesel fuel, etc. can be produced at low cost. it can.
- Example 1 Materials and methods 1-1. Experimental Materials The seeds of Arabidopsis mutants (Activation-tag T-DNA lines: Weigel T-DNS lines, total 20072 lines) were used as experimental materials. The seeds were purchased from Nottingham Arabidopsis Stock Center (NASC). For seeds used as experimental materials, Weigel, D., et al., Plant Physiol., 122, 1003-1013 (2000) can be referred to.
- NSC Nottingham Arabidopsis Stock Center
- T-DNA insertion site into the genome of the selected salt-tolerant Arabidopsis thaliana strain was determined by the TAIL-PCR method. First, young leaves were collected from cultivated Arabidopsis thaliana and ground under liquid nitrogen freezing. DNA was prepared using a DNA preparation kit (DNeasy Plant Mini Kit) manufactured by QIAGEN according to the standard protocol attached to the kit.
- Three kinds of specific primers TL1, TL2 and TL3 are set in the vicinity.
- TAIL-PCR (supervised by Isao Shimamoto, Takuji Sasaki, new edition, plant PCR) using the optional primer P1 under the following PCR reaction solution and reaction conditions Experimental protocol, 2000, 83-89pp, Shujunsha, Tokyo, Genomics, 25, 674-681, 1995, Plant J., 8, 457-463, 1995) to amplify genomic DNA adjacent to T-DNA did.
- primers TL1, TL2, TL3 and P1 are as follows.
- TL1 5'-TGC TTT CGC CAT TAA ATA GCG ACG G-3 '(SEQ ID NO: 6)
- TL2 5'-CGC TGC GGA CAT CTA CAT TTT TG-3 '(SEQ ID NO: 7)
- TL3 5'-TCC CGG ACA TGA AGC CAT TTA C-3 '(SEQ ID NO: 8)
- P1 5'-NGT CGA SWG ANA WGA A-3 '(SEQ ID NO: 9)
- n represents a, g, c, or t (location: 1 and 11)
- s represents g or c (location: 7)
- w represents a or t is represented (location: 8 and 13).
- the first PCR reaction solution composition and reaction conditions are shown in Table 1 and Table 2, respectively.
- Tables 5 and 6 show the composition and reaction conditions of the third PCR reaction solution, respectively.
- the amplification product of the third round was directly sequenced with BigDyeDTerminator Cycle Sequencing Kit Ver.3.1 (Applied Biosystems) using the specific primer TL3, and ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) ) To determine the nucleotide sequence.
- TAIR The Arabidopsis Information Resource
- AGI The abArabidopsis Genome Initiative gene code
- AGI The Arabidopsis Genome Initiative gene code
- At1g70000 the gene of Arabidopsis chromosome 5 [AGI (The Arabidopsis Genome code) : At5g39400]
- Arabidopsis chromosome 3 gene Arabidopsis chromosome 3 gene
- Arabidopsis chromosome 2 gene Arabidopsis Genome Initiative gene code: At2g33110]
- LRR-RLK leucine-rich repeat receptor-like protein kinase gene
- a pair of primers was designed and synthesized based on the sequence information published in home.html) (Table 7). The pair of primers was designed to add a restriction enzyme site necessary for introduction into an expression vector (Table 7).
- Template DNA was prepared from wild-type Arabidopsis thaliana, Col-0 ecotype, according to the method described. Takara Ex Taq (manufactured by Takara Bio Inc.), Platinum Pfx DNA Polymerase (manufactured by Invitrogen) or Phusion High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs: NEB Corp.), and a pair of primers shown in Table 7 as primers Using. The PCR reaction solution composition and reaction conditions followed the protocol attached to each enzyme.
- PCR was performed using the three sets of primers shown in Table 8 and Platinum ⁇ Pfx DNA Polymerase (manufactured by Invitrogen) as an enzyme to obtain three PCR amplification products. .
- Each PCR amplification product was electrophoresed on a 2% agarose gel (TAE buffer), and the fragment was stained with ethidium bromide.
- TAE buffer 2% agarose gel
- the gel containing the target fragment was excised with a scalpel, and the target DNA fragment was eluted and purified using GFX PCR DNA and GEL Purification Kit (Amersham).
- Overlapping PCR was performed using the obtained three DNA fragments as templates and Forward1 and Reverse3 as primers.
- Each PCR amplification product was cut out and purified after electrophoresis on an agarose gel in the same manner as described above.
- Adenine was added to the obtained DNA fragment using A-Addition Kit (manufactured by QIAGEN).
- Amplified DNA added with adenine was ligated to the pCR2.1 vector for TA-Cloning using TOPO TA Cloning Kit (Invitrogen) and transformed into competent cells (E. coli TOP 10) attached to the kit. .
- the cells were cultured in LB medium supplemented with 50 ⁇ l / ml kanamycin, and transformants were selected. The emerged colonies were liquid-cultured in LB medium supplemented with 50 ⁇ l / ml kanamycin, and plasmid DNA was prepared from the obtained cells using Plasmid Mini Mini Kit manufactured by QIAGEN®.
- Vector obtained by cloning an ORF fragment of the obtained LRR-RLK gene (AT1G69990), vector obtained by cloning an ORF fragment of the LRR-RLK gene (AT5G39390), and an LRR (fragment containing an ORF of protein gene (AT3G05650))
- the cloned vector, the vector that cloned the ORF fragment of the LRR protein gene (AT2G33080), and the vector that cloned the CC-NBS-LRR protein gene (AT1G58602) ORF fragment were sequenced and sequenced, respectively. .
- LBI-RLK gene (AT1G69990), LRR-RLK gene (AT5G39390), LRR protein gene (AT3G05650), LRR protein gene (AT2G33080) were added to the plant expression vector pBI121 containing an omega sequence derived from tobacco mosaic virus. ), A construct in which a fragment containing the ORF of the CC-NBS-LRR protein gene (AT1G58602) was inserted was prepared.
- pBI121 containing the omega sequence was similarly treated with restriction enzymes SalSI and BsrG I. These restriction enzyme digests were subjected to 0.8% agarose gel electrophoresis, and fragments of about 1850 bp LRR-RLK gene (AT1G69990) ORF sequence and omega sequence using GFX PCR DNA GEL Band Purification Kit (Amersham) PBI121 containing was fractionated and purified.
- the mixture was mixed at a vector: insert ratio of 1:10, and equal amounts of TaKaRa Ligation Kit ver.
- the ligation reaction was performed overnight at 16 ° C. using 2 (Takara Bio Inc.).
- the total amount of the reaction solution was added to 100 ⁇ l of competent cells (E. coli strain DH5 ⁇ , TOYOBO), and transformation was performed according to the protocol attached to the kit. Apply to LB agar medium containing 50 ⁇ g / ml kanamycin and culture overnight. Appearance colonies are liquid-cultured in LB medium supplemented with 50 ⁇ g / ml kanamycin. From the obtained cells, Plasmid Mini Kit (manufactured by QIAGEN) ) was used to prepare plasmid DNA.
- Plasmid Mini Kit manufactured by QIAGEN
- the primers used were incorporated into an expression vector according to the method described above except that the primers listed in Table 7 were used. Analysis was performed.
- the LRR protein gene (AT3G05650)
- after cloning into the pCR2.1 vector for TA-Cloning treatment with the restriction enzyme EcoR I, blunting with DNA Blunting Kit (manufactured by Takara Bio Inc.), treatment with phenol chloroform, Treated with restriction enzyme BsrG I.
- pBI121 containing the omega sequence was treated with restriction enzyme SalSI, blunt-ended using DNA Blunting Kit (manufactured by Takara Bio Inc.), treated with phenol chloroform, and then treated with restriction enzyme BsrG I.
- the DNA was incorporated into an expression vector according to the method described above, and nucleotide sequence determination and sequence analysis were performed.
- the CC-NBS-LRR protein gene (AT1G58602) was treated with the restriction enzyme Not I, blunt-ended using DNA Blunting Kit (manufactured by Takara Bio Inc.), treated with phenol chloroform, and then treated with the restriction enzyme Sal I.
- pBI121 containing an omega sequence was treated with restriction enzyme BsrG I, then blunt-ended using DNA Blunting Kit (Takara Bio), treated with phenol chloroform, and then treated with restriction enzyme Sal I.
- the DNA was incorporated into an expression vector according to the method described above, and nucleotide sequence determination and sequence analysis were performed.
- a transformant was selected with a kanamycin-containing medium, and a T2 generation plant was produced by self-pollination.
- Biomass measurement : 1-2-7 Each T2 seed prepared in the above was aseptically sown on an MS agar medium containing 50 mg / L kanamycin and 0.5% sucrose, and transplanted to a 50 mm diameter pot containing vermiculite mixed soil two weeks later. Subjects were transplanted with non-recombinant Arabidopsis aseptically seeded on MS agar medium containing 0.5% sucrose. This 23 ° C., 8 h photoperiod 16 hours dark (short-day conditions), and cultivated at a light intensity of about 160 ⁇ E / cm 2, and cultivated a total of 6 weeks after implantation. After cultivation, the above-ground plant body was put in a paper bag and dried for 2 weeks under conditions of 22 ° C. and 60% humidity, and then the total biomass was weighed with an electronic balance.
- FIG. 3 shows a photograph of the above-ground part of a transformed plant into which a wild-type and CC-NBS-LRR protein gene (AT1G58602) ORF-containing fragment was introduced as a result of the biomass amount measurement.
- Fig. 5 shows the results of measuring the total biomass of the above-ground parts for transformed plants, transformed plants introduced with the LRR protein gene (AT2G33080) and transformed plants introduced with the CC-NBS-LRR protein gene (AT1G58602). 4 shows.
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Abstract
Description
(b)配列番号5に示すアミノ酸配列において1又は複数個のアミノ酸が欠失、置換、付加又は挿入されたアミノ酸配列を含み、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質
(c)配列番号4に示す塩基配列の相補的な塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質
また、本発明において、上記機能的に等価な遺伝子は、シロイヌナズナ以外の生物由来のコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子を挙げることができる。シロイヌナズナ以外の生物としては、ブドウを挙げることができる。
本発明において、CC-NBS-LRR遺伝子は、配列番号3に示すアミノ酸配列からなる共通配列及び配列番号2に示すアミノ酸配列からなる共通配列をN末端側からこの順で有する、CC-NBS-LRRタンパク質をコードする。なお、CC-NBS-LRRは、参考文献(1)(The Plant Cell , Vol.15, 809-834, April 2003)に記載されているように、核酸結合部位を有するとともに植物における抵抗性タンパク質(Rタンパク質とも称される)の一種として分類されている。特に、上記共通配列を有するCC-NBS-LRRは、上記参考文献において“CNL-D”として分類されており、N末端側からコイルドコイル構造、核酸結合部位及びロイシンリッチリピート構造を有する点では他のCC-NBS-LRRと同じであるが、コイルドコイル構造とロイシンリッチリピート構造との間における核酸結合性モチーフの一部が特徴的であり、他のCC-NBS-LRRとは異なる生物学的機能を有していると考えられる。
このグループは、上記参考文献(1)で示された中性非極性アミノ酸のうち、脂肪属性の疎水性側鎖をもつアミノ酸のグループであり、V(Val、バリン)、L(Leu、ロイシン)、I(Ile、イソロイシン)及びM(Met、メチオニン)から構成される。参考文献(1)による中性非極性アミノ酸と分類されるもののうちFGACWPは以下理由で、この「脂肪族疎水性アミノ酸グループ」には含めない。G(Gly、グリシン)やA(Ala、アラニン)はメチル基以下の大きさで非極性の効果が弱いからである。C(Cys、システイン)はS-S結合に重要な役目を担う場合があり、また、酸素原子や窒素原子と水素結合を形成する特性があるからである。F(Phe、フェニルアラニン)やW(Trp、トリプトファン)は側鎖がとりわけ大きな分子量をもち、かつ、芳香族の効果が強いからである。P(Pro、プロリン)はイミノ酸効果が強く、ポリペプチドの主鎖の角度を固定してしまうからである。
このグループは、中性極性アミノ酸のうちヒドロキシメチレン基を側鎖に持つアミノ酸のグループであり、S(Ser、セリン)とT(Thr、スレオニン)から構成される。SとTの側鎖に存在する水酸基は、糖の結合部位であるため、あるポリペプチド(タンパク質)が特定の活性を持つために重要な部位である場合が多い。
このグループは、酸性であるカルボキシル基を側鎖に持つアミノ酸のグループであり、D(Asp、アスパラギン酸)とE(Glu、グルタミン酸)から構成される。
このグループは、塩基性アミノ酸のグループであり、K(Lys、リジン)とR(Arg、アルギニン)から構成される。これらKとRは、pHの広い範囲で正に帯電し塩基性の性質をもつ。一方、塩基性アミノ酸に分類されるH(His、ヒスチジン)はpH7においてほとんどイオン化されないので、このグループには分類されない。
このグループは、全てα位の炭素元素に側鎖としてメチレン基が結合しその先に極性基を有すると言う特徴を持つ。非極性基であるメチレン基の物理的大きさが酷似している特徴を持ち、N(Asn、アスパラギン、極性基はアミド基)、D(Asp、アスパラギン酸、極性基はカルボキシル基)とH(His、ヒスチジン、極性基はイミダゾール基)から成る。
6)ジメチレン基=極性基(EKQRグループ)
このグループは、全てα位の炭素元素に側鎖としてジメチレン基以上の直鎖炭化水素が結合しその先に極性基を有すると言う特徴を持つ。非極性基であるジメチレン基の物理的大きさが酷似している特徴を持つ。E(Glu、グルタミン酸、極性基はカルボキシル基)、K(Lys、リジン、極性基はアミノ基)、Q(Gln、グルタミン、極性基はアミド基)、R(Arg、アルギニン、極性基はイミノ基とアミノ基)から成る。
このグループには、側鎖にベンゼン核を持つ芳香族アミノ酸であり、芳香族特有の化学的性質を特徴とする。F(Phe、フェニルアラニン)、Y(Tyr、チロシン)、W(Trp、トリプトファン)から成る。
このグループには、側鎖に環状構造を持つと同時に極性も持つアミノ酸で、H(H、ヒスチジン、環状構造と極性基は共にイミダゾール基)、Y(Tyr、チロシン、環状構造はベンゼン核で極性基は水酸基)から成る。
発現ベクターは、植物体内で発現を可能とするプロモーターと、上述したCC-NBS-LRR遺伝子とを含むように構築する。発現ベクターの母体となるベクターとしては、従来公知の種々のベクターを用いることができる。例えば、プラスミド、ファージ、またはコスミド等を用いることができ、導入される植物細胞や導入方法に応じて適宜選択することができる。具体的には、例えば、pBR322、pBR325、pUC19、pUC119、pBluescript、pBluescriptSK、pBI系のベクター等を挙げることができる。特に、植物体へのベクターの導入法がアグロバクテリウムを用いる方法である場合には、pBI系のバイナリーベクターを用いることが好ましい。pBI系のバイナリーベクターとしては、具体的には、例えば、pBIG、pBIN19、pBI101、pBI121、pBI221等を挙げることができる。
上述した発現ベクターは、一般的な形質転換方法によって対象の植物内に導入される。発現ベクターを植物細胞に導入する方法(形質転換方法)は特に限定されるものではなく、植物細胞に応じた適切な従来公知の方法を用いることができる。具体的には、例えば、アグロバクテリウムを用いる方法や直接植物細胞に導入する方法を用いることができる。アグロバクテリウムを用いる方法としては、例えば、Bechtold, E., Ellis, J. and Pelletier, G. (1993) In Planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis plants. C.R. Acad. Sci. Paris Sci. Vie, 316, 1194-1199. あるいは、Zyprian E, Kado Cl, Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid. Plant Molecular Biology, 1990, 15(2), 245-256.に記載された方法を用いることができる。
ウルシ科:ハゼノキ(Rhus succedanea)、カシューナットノキ(Anacardium occidentale)、ウルシ(Toxicodendron vernicifluum)、マンゴー(Mangifera indica)、ピスタチオ(Pistacia vera)
ウリ科:カボチャ(Cucurbita maxima、Cucurbita moschata、Cucurbita pepo)、キュウリ(Cucumis sativus)、カラスウリ(Trichosanthes cucumeroides)、ヒョウタン(Lagenaria siceraria var. gourda)
バラ科:アーモンド(Amygdalus communis)、バラ(Rosa)、イチゴ(Fragaria)、サクラ(Prunus)、リンゴ(Malus pumila var. domestica)など。
イネ科:トウモロコシ(Zea mays)、イネ(Oryza sativa)、オオムギ(Hordeum vulgare)、コムギ(Triticum aestivum)、タケ(Phyllostachys)、サトウキビ(Saccharum officinarum)、ネピアグラス(Pennisetum pupureum)、エリアンサス(Erianthus ravenae)、ミスキャンタス(ススキ)(Miscanthus virgatum)、ソルガム(Sorghum)スイッチグラス(Panicum)など。
上述した形質転換処理後、植物体のなかから適切な形質転換体を選抜する選抜工程を、従来公知の方法で行うことができる。選抜の方法は特に限定されるものではなく、例えば、ハイグロマイシン耐性等の薬剤耐性を基準として選抜してもよいし、形質転換体を育成した後に、植物体そのもの、または任意の器官や組織の重量を測定して野生型と比較して有意に増産しているものを選抜してもよい。
1.材料および方法
1-1.実験材料
実験材料にはシロイヌナズナ変異体(Activation-tag T-DNA lines: Weigel T-DNS lines、計20072系統)の種子を用いた。なお、種子はNottingham Arabidopsis Stock Centre(NASC) より購入した。実験材料として使用した種子についてはWeigel, D., et al., Plant Physiol., 122, 1003-1013 (2000)を参考とすることができる。
1-2-1.塩耐性変異体の選抜
Weigel T-DNA linesの種子を、NaCl 125 mMあるいは150 mMを含む改変MS寒天(1%)培地〔B5培地のビタミン、ショ糖10 g/l、寒天(細菌培地用;和光純薬工業社製)8 g/L〕に無菌播種し、22℃、30~100μmol/m2/secの照明下(16時間明期/8時間暗期のサイクル)で培養した。播種後2~4週間後、塩耐性変異体候補を選抜した。なお、MS培地はMurashige, T. et al. (1962) Physiol. Plant., 15, 473-497を参照する。また、B5培地についてはGamborg, O.L. et al. (1968) Experimental Cell Research, 50, 151-158を参照する。
選抜した耐塩性シロイヌナズナ系統のゲノムへのT-DNA挿入部位を、TAIL-PCR法により決定した。まず、栽培したシロイヌナズナから幼葉を採取し、液体窒素凍結下で粉砕した。QIAGEN社製DNA調製キット(DNeasy Plant Mini Kit)を用いてキット添付の標準プロトコルに従ってDNAを調製した。
Weigel T-DNA linesで用いられているアクティベーションタギング用ベクター(pSKI015 : GenBank accession No.AF187951)の左のT-DNA配列(T-DNA left border)付近に3種類の特異的プライマーTL1、TL2及びTL3を設定し、任意プライマーP1を用いて、以下のPCR反応液及び反応条件下でTAIL-PCR(島本功、佐々木卓治監修, 新版, 植物のPCR実験プロトコル, 2000, 83-89pp, 秀潤社, 東京、 Genomics, 25, 674-681, 1995、 Plant J., 8, 457-463, 1995)を行い、T-DNAに隣接するゲノムDNAを増幅した。
TL2: 5'-CGC TGC GGA CAT CTA CAT TTT TG-3‘(配列番号7)
TL3: 5'-TCC CGG ACA TGA AGC CAT TTA C-3'(配列番号8)
P1: 5'-NGT CGA SWG ANA WGA A-3‘(配列番号9)
なお、配列番号9において、nは、a、g、c又はtを表し(存在位置:1及び11)、またsは、g又はcを表し(存在位置:7)、さらにwは、a又はtを表す(存在位置:8及び13)。
アクティベートされている遺伝子を、1-2-3.により判明した各々のT-DNA挿入部位(At1g69990とAt1g70000間、At5g39400、At3g05630、At2g33110及びAt1g58520)近傍10Kb以内に存在する推定Open reading frame(ORF)遺伝子の配列から予測した。
1-2-4.でアクティベート(活性化)されていると予測した、LRR-RLK(leucine-rich repeat receptor-like protein kinase )遺伝子 (AT1G69990)、LRR-RLK(leucine-rich repeat receptor-like protein kinase)遺伝子 (AT5G39390)、LRR(leucine- rich repeat)タンパク質遺伝子(AT3G05650)及びLRR(leucine-rich repeat)タンパク質遺伝子(AT2G33080)のORF領域を含む断片を増幅するために、TAIR(http://www.arabidopsis.org/home.html)で公開されている配列情報を基にしてそれぞれ一対のプライマーを設計・合成した(表7)。なお、これら一対のプライマーには、発現ベクターへ導入するときに必要となる制限酵素サイトを付加するように設計した(表7)。
タバコモザイクウイルス由来のomega配列を含む植物発現用ベクターpBI121に、LRR-RLK遺伝子 (AT1G69990)、LRR-RLK遺伝子 (AT5G39390)、LRRタンパク質遺伝子(AT3G05650)、LRRタンパク質遺伝子(AT2G33080)、CC-NBS-LRRタンパク質遺伝子(AT1G58602)のORFを含む断片を挿入したコンストラクトの作製を行った。
1-2-6.で作製した各植物発現用ベクターをエレクトロポレーション法(Plant Molecular Biology Mannal, Second Edition , B. G. Stanton A. S. Robbert, Kluwer Acdemic Publishers 1994)により、アグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)C58C1株に導入した。次いで植物発現用ベクターが導入されたアグロバクテリウム・ツメファシエンスを、Cloughらにより記載された浸潤法(Plant J. 16, 735-743, 1998)により、野生型シロイヌナズナ エコタイプCol-0に導入した。
耐塩性試験:
1-2-7.で作製した各々の種子と、対象として非組換えの野生型シロイヌナズナの種子を、150mM NaClを含むMS寒天培地に無菌播種した。これを22℃、16時間明期8時間暗期、光強度約30~45μE/cm2で10日間培養し、塩耐性試験を行った。
1-2-7.で作製した各T2種子を50mg/Lのカナマイシンと0.5%ショ糖を含むMS寒天培地に無菌播種し、2週間後バーミキュライト混合土を入れた直径50mmのポットに移植した。対象には、0.5%ショ糖を含むMS寒天培地に無菌播種した非組換えシロイヌナズナを移植した。これを23℃、8時間明期16時間暗期(短日条件)、光強度約160μE/cm2で栽培し、移植後合計6週間栽培した。栽培後、地上部の植物体を紙袋に入れ、22℃、湿度60%の条件で2週間乾燥させた後に、全バイオマス量を電子天秤で秤量した。
上記1-2-8.の耐塩性試験結果として、野生型及びCC-NBS-LRRタンパク質遺伝子(AT1G58602)のORFを含む断片を導入した形質転換植物体のプレートを撮影した写真を図2に示した。図2から、CC-NBS-LRRタンパク質遺伝子(AT1G58602)のORFを含む断片を導入した形質転換植物体では、高塩濃度培地において発芽、生育しており野生型と比較して耐塩性が向上していることが明らかとなった。
Claims (27)
- 配列番号3に示すアミノ酸配列からなる共通配列及び配列番号2に示すアミノ酸配列からなる共通配列をN末端側からこの順で有する、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子を導入した、又は内在する当該遺伝子の発現制御領域を改変した植物体。
- 上記タンパク質は、配列番号1に示すアミノ酸配列からなる共通配列を、上記配列番号3に示すアミノ酸配列からなる共通配列のN末端側に更に有することを特徴とする請求項1記載の植物体。
- 上記遺伝子は、AT1G59124、AT1G58807、AT1G59218、AT1G58848、AT1G58602、AT1G58410、AT1G58400、AT1G58390、AT1G59620、AT1G59780、AT1G50180、AT1G53350、AT5G43470、AT5G48620、AT5G35450及びAT1G10920からなる群から選ばれる少なくとも1種の遺伝子若しくは当該遺伝子に機能的に等価な遺伝子であることを特徴とする請求項1記載の植物体。
- 上記遺伝子が、以下の(a)~(c)のいずれかのタンパク質をコードすることを特徴とする請求項1記載の植物体。
(a)配列番号5に示すアミノ酸配列を含むタンパク質
(b)配列番号5に示すアミノ酸配列において1又は複数個のアミノ酸が欠失、置換、付加又は挿入されたアミノ酸配列を含み、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質
(c)配列番号4に示す塩基配列の相補的な塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質 - 上記機能的に等価な遺伝子は、シロイヌナズナ以外の生物由来のコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子であることを特徴とする請求項3記載の植物体。
- 上記シロイヌナズナ以外の生物は、ブドウであることを特徴とする請求項5記載の植物体。
- 双子葉植物であることを特徴とする請求項1乃至6いずれか一項記載の植物体。
- アブラナ科植物であることを特徴とする請求項1乃至6いずれか一項記載の植物体。
- シロイヌナズナであることを特徴とする請求項1乃至6いずれか一項記載の植物体。
- 配列番号3に示すアミノ酸配列からなる共通配列及び配列番号2に示すアミノ酸配列からなる共通配列をN末端側からこの順で有する、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子を導入する、又は内在する当該遺伝子の発現制御領域を改変するバイオマス量を増産させ且つ植物体に塩ストレス耐性を付与する方法。
- 上記タンパク質は、配列番号1に示すアミノ酸配列からなる共通配列を、上記配列番号3に示すアミノ酸配列からなる共通配列のN末端側に更に有することを特徴とする請求項10記載の方法。
- 上記遺伝子は、AT1G59124、AT1G58807、AT1G59218、AT1G58848、AT1G58602、AT1G58410、AT1G58400、AT1G58390、AT1G59620、AT1G59780、AT1G50180、AT1G53350、AT5G43470、AT5G48620、AT5G35450及びAT1G10920からなる群から選ばれる少なくとも1種の遺伝子若しくは当該遺伝子に機能的に等価な遺伝子であることを特徴とする請求項10記載の方法。
- 上記遺伝子が、以下の(a)~(c)のいずれかのタンパク質をコードすることを特徴とする請求項10記載の方法。
(a)配列番号5に示すアミノ酸配列を含むタンパク質
(b)配列番号5に示すアミノ酸配列において1又は複数個のアミノ酸が欠失、置換、付加又は挿入されたアミノ酸配列を含み、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質
(c)配列番号4に示す塩基配列の相補的な塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質 - 上記機能的に等価な遺伝子は、シロイヌナズナ以外の生物由来のコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子であることを特徴とする請求項12記載の方法。
- 上記シロイヌナズナ以外の生物は、ブドウであることを特徴とする請求項14記載の方法。
- バイオマスが双子葉植物であることを特徴とする請求項10乃至15いずれか一項記載の方法。
- バイオマスがアブラナ科植物であることを特徴とする請求項10乃至15いずれか一項記載の方法。
- バイオマスがシロイヌナズナであることを特徴とする請求項10乃至15いずれか一項記載の方法。
- 配列番号3に示すアミノ酸配列からなる共通配列及び配列番号2に示すアミノ酸配列からなる共通配列をN末端側からこの順で有する、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子を導入した、又は内在する当該遺伝子の発現制御領域を改変した形質転換植物を準備する工程と、
前記形質転換植物の後代植物のバイオマス量及び塩ストレス耐性を測定し、当該バイオマス量が有意に向上するとともに塩ストレス耐性を示した系統を選抜する工程とを含む、植物体の製造方法。 - 上記タンパク質は、配列番号1に示すアミノ酸配列からなる共通配列を、上記配列番号3に示すアミノ酸配列からなる共通配列のN末端側に更に有することを特徴とする請求項19記載の製造方法。
- 上記遺伝子は、AT1G59124、AT1G58807、AT1G59218、AT1G58848、AT1G58602、AT1G58410、AT1G58400、AT1G58390、AT1G59620、AT1G59780、AT1G50180、AT1G53350、AT5G43470、AT5G48620、AT5G35450及びAT1G10920からなる群から選ばれる少なくとも1種の遺伝子若しくは当該遺伝子に機能的に等価な遺伝子であることを特徴とする請求項19記載の製造方法。
- 上記遺伝子が、以下の(a)~(c)のいずれかのタンパク質をコードすることを特徴とする請求項19記載の製造方法。
(a)配列番号5に示すアミノ酸配列を含むタンパク質
(b)配列番号5に示すアミノ酸配列において1又は複数個のアミノ酸が欠失、置換、付加又は挿入されたアミノ酸配列を含み、コイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質
(c)配列番号4に示す塩基配列の相補的な塩基配列からなるポリヌクレオチドに対してストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質 - 上記機能的に等価な遺伝子は、シロイヌナズナ以外の生物由来のコイルドコイル構造と核酸結合部位とロイシンリッチリピート構造を有するタンパク質をコードする遺伝子であることを特徴とする請求項21記載の製造方法。
- 上記シロイヌナズナ以外の生物は、ブドウであることを特徴とする請求項23記載の製造方法。
- 双子葉植物であることを特徴とする請求項19乃至24いずれか一項記載の製造方法。
- アブラナ科植物であることを特徴とする請求項19乃至24いずれか一項記載の製造方法。
- シロイヌナズナであることを特徴とする請求項19乃至24いずれか一項記載の製造方法。
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US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
CN113416737A (zh) * | 2021-06-01 | 2021-09-21 | 河南科技大学 | 葡萄过氧化氢受体基因及其编码蛋白与应用 |
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US8822758B2 (en) | 2008-09-25 | 2014-09-02 | Toyota Jidosha Kabushiki Kaisha | Gene capable of increasing the production of plant biomass and method for using the same |
US9297020B2 (en) * | 2008-11-11 | 2016-03-29 | Toyota Jidosha Kabushiki Kaisha | Gene for increasing the production of plant biomass and method of use thereof |
JP5454086B2 (ja) * | 2009-10-30 | 2014-03-26 | トヨタ自動車株式会社 | 植物に環境ストレス耐性を付与する遺伝子及びその利用方法 |
PE20121693A1 (es) | 2010-01-22 | 2012-12-01 | Bayer Ip Gmbh | Combinacion de espiromesifeno y abamectina como insecticidas |
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US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
CN113416737A (zh) * | 2021-06-01 | 2021-09-21 | 河南科技大学 | 葡萄过氧化氢受体基因及其编码蛋白与应用 |
CN113416737B (zh) * | 2021-06-01 | 2022-07-15 | 河南科技大学 | 葡萄过氧化氢受体基因及其编码蛋白与应用 |
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JP5672004B2 (ja) | 2015-02-18 |
CN102165063B (zh) | 2013-07-10 |
US20110239330A1 (en) | 2011-09-29 |
BRPI0919120A2 (pt) | 2015-08-18 |
BRPI0919120B1 (pt) | 2018-08-07 |
US8822758B2 (en) | 2014-09-02 |
AU2009297500A1 (en) | 2010-04-01 |
CN102165063A (zh) | 2011-08-24 |
CA2737735C (en) | 2015-05-19 |
CA2737735A1 (en) | 2010-04-01 |
JPWO2010035784A1 (ja) | 2012-02-23 |
AU2009297500B2 (en) | 2013-07-11 |
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