JP2008099637A - Transformed dwarf plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transformed dwarf plant transferred with a plant-dwarfing gene through searching the gene by the Fox hunting system and isolating and identifying the gene. <P>SOLUTION: The transformed dwarf plant expressibly includes a gene encoding (a) a protein composed of an amino acid sequence expressing a specific sequence or (b) a protein having the activity of dwarfing a plant and composed of a specific amino acid sequence wherein one or several amino acids are deleted, substituted or added in the amino acid sequence expressing the specific sequence. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a dwarf transformed plant, a method for producing the same, and a dwarfing method for a plant.

  Conventionally, as one of the methods for analyzing the function of a gene, using a transcription enhancer sequence incorporated in T-DNA, a mutant that has activated transcription of a plant gene has been prepared. An activation tagging method for cloning a transcription-activated gene is used (Non-patent Document 1). Plant dwarfing genes have been found using this method (Patent Document 1). The dwarfing genes are At4g31910, At1g04910, At4g35700, At1g49770 with NCBI (National Center for Biotechnology Information) accession numbers.

  However, there is a problem in using the activation tagging method for comprehensive gene function analysis (analyzing gene functions existing in the genome collectively). For example, in a model plant such as Arabidopsis thaliana, there are two or more genes on average in the genomic region of 10 Kb. However, in the activation tagging method, an enhancer sequence is used as an activator in the tag, and transcriptional activation. Since the possible genomic region extends to 5Kb before and after the insertion site, the effect of gene activation by an enhancer is not limited to one gene, but transcription of multiple genes is activated, resulting in a complex phenotype (non-patented) Reference 2).

In order to avoid this problem, the present inventors have developed a technique named “Fox hunting system (Full length cDNA over-expression gene hunting system)” (Patent Document 2). The Fox hunting system was obtained by using a full-length cDNA library as a gene source for strong expression and introducing the gene into a plant via Agrobacterium having a strong expression T-DNA vector as a mixture. In this method, T ₁ seeds are sown and phenotypic screening is performed. If an interesting phenotype appears, the full-length cDNA inserted into the strain is examined by PCR and sequencing to identify the causative gene.

  Advantages of the Fox hunting system include, for example, (i) the full-length cDNA library contains all amino acid information necessary for the function of the gene, and therefore can exhibit all the functions inherent to the gene to be introduced. Compared to normal cDNA libraries, the function expression efficiency is much higher, and all cDNA fragments have the original start and stop codon information, so there is no need for protein fusion for expression. Ii) High protein expression efficiency; (ii) Even if infected with a library of hundreds of millions of clones, only one or two clones can be introduced into one plant. Isolation and confirmation of phenotypes can be done in less than two times. (Iii) In conventional cDNA libraries, all mRNA molecules are replaced with cDNA molecules in the same quantitative ratio, so that the expression level is high. The cDNA abundance ratios of structural protein genes and usually low-expressing information-related genes differ greatly depending on the expression level, but in the Fox hunting system library, all clones regardless of the gene expression level Can be used in a “standardized” library containing the same percentage, and functional testing of different genes can be performed more efficiently than when tagging genomes (however, standardized full-length cDNAs such as Arabidopsis thaliana and rice You can use it as it is being prepared, or you can use a non-standardized full-length cDNA library if you want to analyze the function of a protein with a high expression level, such as a structural protein. ), (Iv) all the plants in the library without having to “line up” the plant population that contains the full-length cDNA. Gene function search can be performed in a lump so that the target mutant can be easily obtained, and a gene that imparts a specific property with little effort (whether it is a function that the gene originally has or not) Screening) can be performed.

International Publication No. 2005/026345 Pamphlet Republished 2003/018808 Walden, R et al., Plant Mol Biol, 1994, 26 (5): p. 1521-8 Ichikawa et al., Plant J, 2003, 36: p.421-429

  An object of the present invention is to find a plant dwarfing gene and provide a plant transformed with the gene.

  The present inventors made about 15,000 Arabidopsis transformant lines using the Fox hunting system, and observed the mutant phenotype line, and found morphological mutants that showed dwarfing, The present inventors have succeeded in isolating the gene responsible for the mutation and completed the present invention.

That is, the present invention includes the following inventions.
(1) (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, or
(b) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having an activity of miniaturizing a plant can be expressed. A dwarf transformed plant.
(2) The gene is
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1, or
(d) a DNA encoding a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 and has an activity of dwarfing a plant body
The transformed plant according to (1), comprising:
(3) The plant is a dicotyledonous plant or a monocotyledonous plant, for example, one belonging to any family selected from the group consisting of Brassicaceae, Eggplant, Gramineae and Legumes, (1) or (2 The transformed plant according to (1).
(4) The dwarfing according to any one of (1) to (3), which comprises regenerating a plant by introducing the gene defined in (1) or (2) into a plant tissue or cell. A method for producing a transformed plant.
(5) The method according to (4), wherein the gene is introduced using a recombinant vector containing the gene.
(6) The transformed plant according to any one of (1) to (3), which comprises overexpressing the gene defined in (1) or (2) to induce dwarfing Dwarf method.

  According to the present invention, it becomes possible to transform a plant dwarfing gene into various plants to dwarf the plant, which makes it more resistant to wind damage such as typhoons without falling down when the number of grains increases, for example. It is possible to increase the yield of crops, etc., and to diversify phenotypes. In the case of ornamental plants, plants with different silhouettes can be created by shortening flower stems, etc., leading to the creation of new product value.

1. Isolation of Plant Dwarfing Gene Plant dwarfing gene can be obtained by, for example, Fox hunting system using Arabidopsis thaliana. However, the following method is not limited to Arabidopsis thaliana and can be applied to other plants (particularly angiosperms).

Specifically, the following procedure can be used.
(1) Preparation of cDNA mixture containing full-length cDNA and internal standard gene Full-length cDNA means a complete copy of mRNA, and should be included in full-length cDNA even when cDNA longer than the obtained cDNA is present. Can do.

  Full-length cDNA can be prepared from the mRNA of interest by methods known to those skilled in the art. For example, from mRNA derived from Arabidopsis thaliana, Cap-trapper method (Carninci, P., et al., Genomics, 1996. 37 (3): P. 327-36), Cap-finder method (Zhao, Z., et al., J Biotechnol., 1999. 73 (1): p.35-41). Alternatively, the Arabidopsis full-length cDNA collection of RIKEN, where cDNA sequencing has been performed, may be used.

  A vector for cloning a full-length cDNA has a restriction enzyme site on both sides of the cDNA insertion site, such as SfiI, which recognizes 8 bases or more and can define the direction of the inserted DNA in one direction. It is preferable to use it.

  A full-length cDNA library carrying the full-length cDNA can be prepared using techniques known to those skilled in the art. Examples of vectors for preparing the library include Lambda Zap II, Lambda FLC-1-B, pTAS, and pBIG.

Further, as an internal standard for screening phenotype later, for example, a bacterial-derived tumor gene (tms1, iaaM, rolB, tmr, etc.) may be used. tms1 and iaaM are genes encoding tryptophan 2-monooxygenase from Pseudomonas syringae and Agrobacterium tumefaciens , respectively, and are highly expressed when auxin response is enhanced To do. rolB is a gene related to auxin sensitivity derived from Agrobacterium rhyzogenesis . tmr is a gene that functions in cytokinin biosynthesis derived from Agrobacterium tumefaciens. These oncogenes are known to have dramatic effects on the morphology of many plants (Casanova et al., Biotechnol Adv, 23, 2005, 3-39; Romano et al., Plant Mol Biol, 27, 1995, 1071-83; Smart et al., Plant Cell, 3, 1991, 647-656). Oncogenes can be used as an indicator of library cDNA homogeneity, but after transformation into Arabidopsis, these oncogenes are excluded from the mutant lineage in the second generation. These oncogenes can be amplified by PCR.

(2) Standardization of full-length cDNA library As described in the background art, in the conventional cDNA library, the abundance ratio of each cDNA clone varies greatly depending on the expression level of the gene. Therefore, it is preferable to perform “standardization” in which a library is prepared so that all clones are included in the same ratio regardless of the gene expression level.

  A standardized full-length cDNA mixture can be obtained by mixing equal amounts of full-length cDNA for each clone. To do this, determine the 5 'and 3' end sequences of the synthesized full-length cDNA (single-pass sequence), and have no overlap (the sequence of some regions at the end is not common) A long cDNA clone is selected and stored in a database.

  A standardized full-length cDNA library is prepared by selecting different cDNAs and mixing them in equal amounts, and there is no heterogeneity in the molecular species of the conventional cDNA library, and it is uniform overall. . Therefore, when a multi-copy gene group of genomic genes is also taken into consideration, it is possible to perform a functional test of another gene more equitably, that is, with higher efficiency than when tagging a genome.

  In Arabidopsis thaliana, standardized full-length cDNAs corresponding to 50% or more of the entire genome are currently prepared, and these prepared standardized full-length cDNAs can be used in the present invention.

(3) Cloning of full-length cDNA into expression vector Cloning of the obtained full-length cDNA or standardized full-length cDNA into a T-DNA expression vector for plant transformation with Agrobacterium tumefaciens it can. T-DNA is a specific region of the Ti plasmid found in the pathogenic strain of Agrobacterium, the pathogenic bacterium of crown gall, a dicotyledonous tumor, and when this bacterium infects plants, T-DNA Is transferred to plant cells and incorporated into genomic DNA.

This T-DNA contains a sequence for regulating the expression of full-length cDNA. As an expression control sequence, it is preferable to incorporate a promoter sequence that causes expression constitutively or conditionally in a plant cell and a cassette linked with a terminator. Preferred constitutively expressed promoter sequences include the Cauliflower Mosaic Virus 35S promoter sequence (Sanders, PR, et al., Nucleic Acids Res, 1987.15 (4): p.1543-58). Are glucocorticoid-inducible promoter sequences (Aoyama, T. et al., Plant J, 1997.11 (3): p.605-12), estrogen-inducible promoter sequences (Zuo, J et al., Plant J, 2000.24 (2): p. 265-273). In the present invention, these promoters can be used in any combination (linked). The combination of promoters may be a constitutive expression type or an induction type, or a combination of both.

  Further, for example, a hygromycin resistance gene may be inserted for subsequent plant body selection.

  The aforementioned full-length cDNA or standardized full-length cDNA is inserted downstream of the promoter sequence in the sense direction or the antisense direction by an enzymatic reaction, for example, using T4 ligase. As a result, when the sense strand is expressed, the phenotypic change caused by overexpression of the gene encoding the cDNA results, and when the antisense strand is expressed, the gene encoding the cDNA is underexpressed. You can know changes in phenotypes.

(4) Introduction of full-length cDNA library into Arabidopsis thaliana Next, the full-length cDNA or standardized full-length cDNA, or a T-DNA population into which an oncogene is inserted (Full-length cDNA over-expressor library; FOX library ) Is introduced into Agrobacterium by a conventional method to prepare a library, and then the cDNA in the library is introduced (transformed) into Arabidopsis thaliana via infection with Agrobacterium.

  Infecting plants with Agrobacterium can be performed using a dipping method, a floral dipping method, or the like. In the case of the dipping method, the bundle of plants is immersed in a liquid containing Agrobacterium for 30 to 60 seconds. In the case of the floral dipping method, the flower buds of the plant host are directly dipped, the pots are transferred to a tray, covered and kept overnight. Uncover the next day, grow the plants as they are, and harvest the seeds.

(5) seeding seeds obtained Arabidopsis using FOX library screening Agrobacterium by phenotype from the transformed plants transformed (T ₀ generation), the medium containing e.g. hygromycin. Since transformed plants can be selected within about one week of germination, no further cultivation is required to select hygromycin resistant seedlings. The selected plants (T ₁ generation) are transplanted into soil and seeds are collected.

(6) Reconfirmation of expression traits and identification of genes that cause mutation traits Transformed plants of interest (for example, those with slow growth of plants compared to other transformed plants, those with short internodes, Extracting genomic DNA from plants that have traits presumed to be related to dwarfing, such as those showing low pregnancies), and from this DNA, the nucleotide sequence information in the vicinity of the promoter sequence and terminator sequence contained in T-DNA is also obtained. In addition, primers are designed and used to perform polymerase chain reaction (PCR) to isolate cDNA sandwiched between these transcription control regions. This cDNA is again inserted into T-DNA having the same promoter sequence and terminator sequence as described above, and this is reintroduced into a normal plant to reconfirm the phenotype. Then, by sequencing the cDNA, it is possible to identify the gene that causes the mutation.

2. Isolated and identified dwarfing gene The plant dwarfing gene isolated and identified by the above technique is At1g66820 (strain name: F02347) of Arabidopsis thaliana. The base sequence of At1g66820 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In the base sequence of SEQ ID NO: 1, the 1st to 53rd positions are 5'UTR, and the 384th to 544th positions are 3'UTR.

  As long as the protein consisting of each amino acid sequence shown in SEQ ID NO: 2 has the activity of dwarfing plants, a plurality of, preferably one or several amino acids in the amino acid sequence have mutations such as deletion, substitution, addition, etc. May be.

  For example, 1 to 10, 1 to 8, and preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2 may be deleted, added or substituted.

  Further, it may be a gene encoding a protein having 70% or more homology with the amino acid sequence shown in any one of SEQ ID NO: 2 and encoding a protein having an activity of dwarfing a plant body. The homology of 70% or more preferably means 80% or more, 85% or more, more preferably 90% or more, and most preferably 95% or more.

  The amino acid deletion, addition, and substitution can be performed by modifying a gene encoding the protein by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto, for example, a mutation introduction kit (for example, Mutant-K using site-directed mutagenesis). (TAKARA) or Mutant-G (TAKARA)) or the like, or the TAKARA LA PCR in vitro Mutagenesis series kit is used to introduce mutations.

  Here, in the present invention, the “activity for dwarfing a plant” means an activity for growing a plant when a dwarfing gene is expressed more slowly than the wild type. For example, it means an activity of reducing the height of a plant body (eg, Arabidopsis thaliana) when expressing a dwarfing gene to 2/3 to 1/10, preferably 1/3 to 1/10 of the wild type. Further, “having an activity of dwarfing a plant” means that the activity is substantially equivalent to the activity of a protein having the amino acid sequence shown in SEQ ID NO: 2. A plant having such a dwarfing activity (or dwarfing ability) is referred to herein as a dwarfed plant.

  The dwarfing gene also encodes a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 and has an activity of dwarfing a plant. Contains DNA to do. Here, 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. and 6 × SSC (sodium chloride / sodium citrate), followed by washing at 50 to 65 ° C., 0.2 to 1 × SSC, 0.1% SDS, or such conditions , 65-70 ° C., hybridization at 1 × SSC, and subsequent washing at 65-70 ° C., 0.3 × SSC.

  Once the base sequence of the miniaturized gene is determined, it is then reduced by chemical synthesis, by PCR using the cloned cDNA as a template, or by hybridization using a DNA fragment having the base sequence as a probe. Gene can be obtained. Furthermore, modified DNA encoding the dwarfing gene can be synthesized by site-directed mutagenesis or the like.

3. Production of Recombinant Vector and Transformed Plant (1) Production of Recombinant Vector The recombinant vector used in the present invention can be produced by inserting the dwarfing gene into an appropriate vector. As a vector for introducing and expressing a dwarfing gene into a plant cell, a pBI vector, a pUC vector, and a pTRA vector are preferably used. The pBI and pTRA vectors can introduce a target gene into a plant via Agrobacterium. A pBI binary vector or an intermediate vector system is preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) can also be used.

  In order to insert the dwarfing gene into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or multiple cloning site, and ligated to the vector. Is done.

  The dwarfing gene needs to be incorporated into a vector so that the function of the gene is exhibited. Thus, in addition to a promoter and a dwarfing gene, an enhancer, a splicing signal, a poly A addition signal, a selection marker, a 5′-UTR sequence, and the like can be linked to the vector as desired. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, a hygromycin resistance gene, and a bialaphos resistance gene.

  The “promoter” does not have to be derived from a plant as long as it functions in plant cells and can induce expression in a specific tissue of a plant or in a specific developmental stage. Specific examples include a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase gene promoter (Pnos), a corn-derived ubiquitin promoter, a rice-derived actin promoter, a tobacco-derived PR protein promoter, and the like.

  The “terminator” may be any sequence that can terminate the transcription of the gene transcribed by the promoter. Specific examples include nopaline synthase gene terminator (Tnos) and cauliflower mosaic virus poly A terminator.

  The “enhancer” is used to increase the expression efficiency of the target gene. For example, an enhancer region containing an upstream sequence in the CaMV35S promoter is preferable.

(2) Production of Dwarfed Transformed Plant The dwarfed transformed plant of the present invention is obtained by introducing the dwarfed gene or a recombinant vector containing the same into a host so that the target gene can be expressed. Can do. A transformed plant (transgenic plant) can be obtained as follows.

  The subject of transformation in the present invention is a plant tissue (e.g. epidermis, phloem, soft tissue, xylem, vascular bundle, etc., including plant organs (e.g. leaves, petals, stems, roots, seeds, etc.)) or plant cells. is there.

Plants used for transformation include dicotyledonous plants and monocotyledonous plants, for example, plants belonging to the family Brassicaceae, Gramineae, Solanum, Legumes (see below), but are limited to these plants is not.
Brassicaceae: Arabidopsis thaliana , rape, cabbage, brassica, etc.
Solanum : Nicotiana tabacum , eggplant, potato ( Solaneum ), tomato ( Lycopersicon ), capsicum ( Capsicum ), etc.
Rosaceae: Rose ( Rosa ), Strawberry ( Fragaria ), Sakura ( Prunus ), Apple ( Malus ), etc.
Asteraceae: Chrysanthemum , sunflower ( Helianthus ), etc.
Dianthus : Carnation ( Dianthus caryophyllus ).
Gramineae: corn ( Zea mays ), rice ( Oryza sativa ), barley ( Hordeum ), wheat ( Triticum ), etc.
Orchidaceae: Cattleya (Cattleya), such as the moth orchid (Phalaenopsis).
Lily family: Tulipa, etc.
Legumes: Soybean ( Glycine max ), Pea ( Pisum ), Broad bean ( Vicia ), Fuji ( Wisteria ), etc.

  Examples of the method for introducing the dwarfing gene or the recombinant vector into the plant include an Agrobacterium method, a PEG-calcium phosphate method, an electroporation method, a liposome method, a particle gun method, and a microinjection method. For example, when the Agrobacterium method is used, a protoplast may be used or a tissue piece may be used. When using protoplasts, co-culture with Agrobacterium with Ti plasmid, fusing with spheroplasted Agrobacterium (spheroplast method), or when using tissue pieces, use leaf disc for target plant The method can be carried out by infecting a sterile cultured leaf piece (leaf disc method) or infecting callus (undifferentiated cultured cells). In addition, for transformation of monocotyledonous plants by the Agrobacterium method, acetosyringone can be used to increase the transformation rate.

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, in situ hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. After PCR, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band. By doing so, it can confirm that it transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, the amplification product may be bound to a solid phase such as a microplate, and the amplification product may be confirmed by fluorescence or enzymatic reaction.

  Tumor tissue, shoots, hairy roots, and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at an appropriate concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.). Plant regeneration generally does not include hormones after roots are differentiated on a medium mixed with appropriate types of auxin and cytokinin, then transplanted to a medium rich in cytokinin and differentiated into shoots This is done by transplanting to the soil.

  Thus transformed plants into which a dwarfing gene such as At1g66820 has been introduced show a dwarf phenotype. Arabidopsis thaliana introduced with the At1g66820 gene also exhibits a phenotype with short internodes.

  In this specification, the term “phenotype” is used synonymously with “phenotype”. These refer to plant morphological features that can be easily observed or measured.

  The dwarf phenotype has the advantage of reducing the possibility of plants falling even when exposed to damage (wind damage, etc.) caused by natural environments such as typhoons. In addition, it is possible to provide a small plant that suits the consumer's preference as an ornamental plant.

4). Plant Dwarfing Method According to the present invention, there is provided a plant dwarfing method comprising inducing a dwarfing in a transformed plant by overexpressing a plant dwarfing gene.

  Methods for overexpressing the dwarfing gene include, for example, substance administration and environmental stress application to growing plants. In the case of an inducible promoter, in the case of a stress-inducible promoter such as administration of a chemical substance (steroid, alcohol, etc.) or temperature, drying, etc., physical stress corresponding to each becomes an inducing condition.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.
1. Method (1) Preparation of cDNA mixture composed of standardized full-length cDNA and marker gene An Arabidopsis thaliana Columbia full-length cDNA library was constructed using the following two vectors. The library RAFL2 to RAFL6 (RAFL: RIKEN Arabidopsis full-length cDNA clone) contains the vector Lambda Zap II (Stratagene, La Jolla, USA), and the library RAFL7 to RAFL11 contains the vector Lambda FLC-1-B (Stratagene, La Jolla, USA).

  Single-pass sequencing clones from these libraries (Seki et al., 2002, Plant J, 31, 279-92) to select non-overlapping clones and approximately 15,000 cDNA with an average final concentration of 14 ng / ml for each clone A full-length cDNA mixture consisting of was prepared.

  About one-third of these clones contain certain duplicated cDNAs that RAFL clone sequencing project (Yamada et al., 2003, Science 31 October 2003: Vol. 302.no. 5646, pp. 842-846) This clone mix will consist of approximately 10,000 non-overlapping full-length cDNAs.

The orientation of the full length cDNA relative to the SfiI cloning site in the RAFL2 and RAFL3 libraries (corresponding to a total of 1,623 clones) was opposite to the remaining SfiI cloning site of the RAFL clone. Auxin synthesis (tms1, iaaM) (Comai et al., 1982, J Bacteriol, 149, 40-6; Klee et al., 1984, Proc Natl Acad Sci USA, 81, 1728- 32), 4 related to auxin sensitivity (rolB) (Furner et al., 1986, Nature, 319, 422-427) and cytokine synthesis (tmr) (Lichtenstein et al., 1984, J Nol Appl Genet, 2, 354-62). Two bacterial genes were amplified by PCR and cloned into the SfiI site of the pBluescript-derived vector. Each control plasmid was added separately to the full-length cDNA mixture at a concentration of 30 ng / ml. The DNA template and PCR primer of the control gene used for amplification are as follows.
pT281 (tms1):
5'-AGAGGCCAAATCGGCCATGTCAGCTTCACCTCTCCTT-3 '(SEQ ID NO: 3)
5'-AGAGGCCCTTATGGCCCTAATTTCTAGTGCGGTAGTTAT-3 '(SEQ ID NO: 4)
pCP3 (iaaM):
5'-AGAGGCCAAATCGGCCATGTATGACCATTTTAATTCACCC-3 '(SEQ ID NO: 5)
5'-AGAGGCCCTTATGGCCCTAATAGCGATAGGAGGCGTTG-3 '(SEQ ID NO: 6)
pLJ-1 (rolB):
5'-TCCTCTAGAGGCCAAATCGGCCATGGATCCCAAATTGCTATTCCT-3 '(SEQ ID NO: 7)
5'-TGATCTAGAGGCCCTTATGGCCTTAGGCTTCTTTCTTCAGGTTTA-3 '(SEQ ID NO: 8)
pT281 (tmr):
5'-AGAGGCCAAATCGGCCATGGACCTGCATCTAATTTTCG-3 '(SEQ ID NO: 9)
5'-AGAGGCCCTTATGGCCCCTAATACATTCCGAACGGATGA-3 '(SEQ ID NO: 10)

(2) Preparation of standardized full-length cDNA Agrobacterium library The cDNA mixture prepared in (1) above was digested with SfiI (Takara Bio), and T4 ligase (New England BioLabs) was added to the SfiI site of the Agrobacterium binary vector pBIG2113SF. , Beverly, USA). The pBIG2113SF vector is derived from pBIG2113N (Taji et al., 2002, Plant J, 29, 417-26), with two SfiI sites in the XbaI site of pBIG2113N so that the full-length cDNA is inserted in the sense orientation relative to the 35S promoter. Inserted.

  FIG. 1 (a) shows an example of a binary vector. El is the 5'-upstream sequence of the CaMV 35S promoter (-419 to -90), P35S is the CaMV 35S promoter (-90 to -1), Ω is the 5'-upstream sequence of TMV, NOS-T is the nopaline of Ti plasmid The polyadenylation signal of the synthetic gene, Hyg, indicates a hygromycin resistance gene. The arrows indicate the position of the GS4 and GS6 primers used to recover the cDNA. LB indicates a left border and RB indicates a right border.

  Ligation was assembled with a 6-fold molar excess of pBluescript over the binary vector, E. coli DH10B (Invitrogen) was transformed with the ligation product by electroporation, colonies were mixed, and the plasmid library was isolated. did.

  Agrobacterium GV3101 was transformed by electroporation with a plasmid library, and the resulting bacterial colonies were mixed to prepare an Agrobacterium library.

(3) Transformation and plant growth Transformation and plant growth are described in a previous report (Ichikawa et al., 2003, Plant J, 36, 421-429).

  Short wild-type Arabidopsis thaliana (Col-0) and transformants were grown at 22 ° C under long-day conditions (16 hours light period, 8 hours dark period) in a cultivation container system (Arasystem, Gent, Belgium). Wild type plants were transformed by the floral dipping method (Clough et al., 1998, Plant J, 16, 735-43) using an Agrobacterium library.

Hygromycin resistance T ₁ seedlings were selected on basic agar medium (BAM) 7 days containing 50 mg / l hygromycin, transferred to soil (Nakazawa et al., 2003, Biotechniques, 34, 28-30 ). Visible phenotypes (eg, growth rate, plant color, flowering period, fertility change, etc.) were scored and all plants exhibiting the phenotype (FT1P) were transferred to a new Arasystem tray for further observation.
For DNA analysis, either rosette leaves or flowers were collected from all FT1P plants.

(4) DNA gel blot analysis and hygromycin resistance test Southern blotting was performed according to a previous report (Meyer et al., 1995, Mol Gen Genet, 249, 265-73). Twenty lines were randomly selected and T ₂ plants were grown for 3 weeks as described in (3) Plant material and plant growth. Leaves were collected from 3 plants per line and homogenized in liquid nitrogen.

  Genomic DNA was isolated using DNeasy plant mini kit (Qiagen, Tokyo, Japan) according to the instructions. A 0.5 kb PCR fragment amplified from pBIG2113SF containing the hygromycin resistance gene portion was labeled with DIG DNA Labeling Mix (F. Hoffmann-La Roche, Basel, Switzerland).

The following were used as PCR primers for DNA templates.
HN: 5'-ATGAAAAAGCCTGAACTCACCG-3 '(SEQ ID NO: 11)
HC: 5'-TCGAGAGCCTGCGCGACG-3 '(SEQ ID NO: 12)

  Hybridization was performed using a solution (5XSSC, 50% formamide, 0.1% N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent (F. Hoffmann-La Roche, Basel, Switzerland)) at a probe concentration of 5 ng / ml. The procedure was followed except that the second washing was performed using a solution of 0.1XSSC and 0.1% SDS at 44 ° C.

  Chemiluminescence was detected by LumiVisionPRO (Aisin) after treating the hybridization membrane with DIG Nucleic Acid Detection Kit (F. Hoffmann-La Roche, Basel, Switzerland).

  To examine hygromycin resistance, an average of 40 seeds obtained from the FT1P line were sown in a hygromycin-containing BAM medium. Ten days after germination, the number of resistant seedlings per seed sowed was counted.

(5) Amplification and cloning of full-length cDNA from FT1P plant Approximately 200 mg (biomass) of rosette leaves or flowers were collected.
Five ceramic grains (CERAMICS YTZ ball, D: 2.3 mm, Nikkato, Japan) and 300 μl lysis buffer are added to the plant material and homogenized using Shake Master (Shake Master ver.1.0, Bio Medical Science, Tokyo, Japan). did. Genomic DNA was extracted using the Wizard Magnetic 96 DNA Plant System (Promega, Tokyo, Japan). A workstation system (Tecan genesis workstation 150, Tecan, Tokyo, Japan) was introduced to carry out the extraction protocol described in the extraction kit.

When cloning into the Gateway vector, the following primers were used for cDNA PCR.
B1GS7: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCTAGAGGCCCTTATGGCCG-3 '(SEQ ID NO: 13)
B2GS8: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTCGAGTTAATTAAATTAATCCCCC-3 '(SEQ ID NO: 14)

The following primer pairs were used for cloning into the pBIG2113SF vector:
GS4: 5'-ACATTCTACAACTACATCTAGAGG-3 '(SEQ ID NO: 15)
GS6: 5'-CGGCCGCCCCGGGGATC-3 '(SEQ ID NO: 16)

  PCR conditions for short fragments were 94 ° C for 30 seconds denaturation, 62 ° C for 30 seconds annealing, 72 ° C for 120 seconds extension. The PCR conditions for the long fragment were 94 ° C for 30 seconds denaturation, 58 ° C for 30 seconds annealing, and 68 ° C for 180 seconds extension. In both cases, the DNA was denatured at 95 ° C. for 8 minutes before the reaction.

(6) Cloning and sequencing of PCR fragments into expression vectors
The Gateway vector PCR fragment was first cloned into the pDONR-207 vector with BP clonase (Invitrogen, Carlsbad, USA) according to the instructions, and the inserted full-length cDNA fragment was sequenced using the following attL1 and attL2 primers.
attL1: 5'-TCGCGTTAACGCTAGCATGGATCTC-3 '(SEQ ID NO: 17)
attL2: 5'-GTAACATCAGAGATTTTGAGACAC-3 '(SEQ ID NO: 18)

for cloning into PBIG2113SF, the PCR fragment from T ₁ plants was digested with SfiI, and cloned into SfiI site of the vector. For example, in the case of the F02347 strain, the obtained construct was designated as pF02347. The inserted full length cDNA fragment was sequenced using GS6 primer.

(7) Comparison of wild type plant and dwarfing gene transformed plant
Genomic DNA was extracted from plants of the F02347 strain, cDNA was amplified as described above using GS4 and GS6 primers, and cloned into pBIG2113SF. Using this construct and Agrobacterium, it was introduced into wild-type Arabidopsis thaliana to produce a transformant of At1g66820 gene. One transformant was selected, the seed was recovered, and 32 offspring thereof were grown, and the height of the plant was measured one month after sowing. Furthermore, seeds were collected from each of these 32 individuals, seeded in a hygromycin medium, nine of the 32 individuals that had not been inserted with T-DNA were identified, and 23 individuals that had been inserted with T-DNA, A comparison was made of the plant lengths of nine individuals that were not.

2. Results (1) Construction of an Agrobacterium expression library containing about 10,000 standardized Arabidopsis full-length cDNAs About 10,000 independent Arabidopsis full-length cDNAs were mixed in the same molar ratio as to create an Arabidopsis full-length cDNA library. The cDNA is derived from Riken's Arabidopsis full-length cDNA collection, and each cDNA has been sequenced (Seki et al., 2002, Plant J., 31, 279-92). Also, as an internal standard, four bacterial oncogenes known to induce morphological phenotype and sterility were mixed. This internal standard can be used as an indicator of library cDNA expression, but after transformation into Arabidopsis, the oncogene is excluded from the mutant line in the second generation. tms1 (Klee et al., 1984, Proc Natl Acad Sci USA, 81, 1728-32), iaaM (Comai et al., 1982, J Bacteriol, 149, 40-6), rolB (Furner et al., 1986, Nature, 319, 422- 427) and tmr (Lichtenstein et al., 1984, J Mol Appl Genet, 2, 354-62). These oncogenes are known to have dramatic effects on the morphology of many plants. When these four oncogenes are added in an amount of about twice the molar ratio to each full-length cDNA, it is predicted that each oncogene will appear once for every 7,500 cDNA clones.

  After preparing an Agrobacterium library (FOX Agrobacterium library), T-DNA specific primers were designed, and PCR was performed with DNA isolated from randomly selected Agrobacterium colonies. PCR fragments obtained from 34 Agrobacterium colonies were amplified and subjected to electrophoresis. As a result, 20 colonies contained single-stranded cDNA constructs, 11 colonies contained two different cDNA constructs, and one colony differed from three. Two colonies contained the cDNA construct and an empty vector (FIG. 1 (b)). In FIG. 1 (b), lanes 1, 2, 5, 6, 7, 9, 10, 13, 15, 17, 19, and 22 showed that multiple bands were amplified (lane M was Lambda / HindIII marker).

  In addition, the average size of these 45 amplified cDNA fragments was 1.4 Kb, which was in the range of 0.3 Kb to 3.0 Kb. When 45 cDNA fragments were sequenced, all cDNAs were independent of each other.

(2) Production of Arabidopsis FOX strain To produce Arabidopsis FOX strain, Arabidopsis Columbia-0 (Col-0) ecotype was transformed using FOX Agrobacterium library. After floral dipping method, collect seeds obtained from T ₀ plants were selected on hygromycin-containing BAM plates (Nakazawa et al., 2003, Biotechniques, 34, 28-30 ). Since the transformed plants are selected within one week of germination by the plate, there is no need to further culture to select hygromycin resistant seedlings. Hygromycin-resistant T ₁ seedlings were transplanted into soil to produce over 15,000 Arabidopsis fertile FOX lines. To confirm the presence of the transgene, 24 randomly selected plants were analyzed by the Southern method for the presence of the marker gene using the hygromycin gene as a probe. All strains contained the hygromycin resistance gene, and an average of 2.6 T-DNA was inserted into the Arabidopsis FOX strain.

(3) Size distribution and sequence differences of full-length cDNAs of Arabidopsis FOX strains Full-length cDNAs were incorporated into expression vectors, and the average size distribution was 1.4 Kb and was within the range of 0.3 to 3 Kb. It was investigated whether it was reflected in the Arabidopsis FOX strain.

  To examine the size distribution and variability of cDNA integrated in FOX strains, PCR was performed on genomic DNA isolated from randomly selected FOX plants using T-DNA specific primers. The size distribution in the 106 lines was an average of 1.4 Kb within the range of 0.3 Kb to 4.2 Kb. FIG. 1 (c) shows the size distribution of 106 RAFL cDNA fragments amplified from Arabidopsis FOX plants compared to 277 randomly selected RAFL cDNAs (white bars) in the Lamdba vector. To clarify the similarity of the distribution patterns, the values obtained from the Lambda vector were divided by 2 and plotted on a graph.

  The average number of PCR fragments amplified from these lines was 1.2 per plant. When DNA gel blot analysis was performed, an average of 2.6 T-DNA was inserted per strain. However, only an average of 1.2 PCR fragments were recovered from FOX plants. This low PCR fragment recovery can be explained in part by the presence of empty vectors in the population, but mainly results in double T-DNA integration such as T-DNA tandem or inverted repeats. (De Buck et al., 1999, Plant J, 20, 295-304; De Neve et al., 1997, Plant J, 11, 15-29; Krizkova et al., 1998, Plant J, 16, 673-80 ).

  The average size and extent of the distribution of cDNA was very similar to that observed with the FOX Agrobacterium library and similar to that observed with 277 randomly selected Arabidopsis full-length cDNAs (FIG. 1 (c)).

  In addition, 40 PCR fragments were sequenced and were derived from different full-length cDNAs and were not identical to the bacterial oncogene added to the cDNA mixture as an internal standard (Table 1).

(4) Monitoring of Arabidopsis FOX strain phenotype
In the process of producing 15,547 T ₁ FOX lines, phenotypes (growth rate, plant color, flowering period, fertility, etc.) were monitored and 1,487 lines with altered phenotypes were collected.

  Compared to these apparent morphological mutant lines and the morphological mutants that appeared in the Arabidopsis activated tactile lines, the distribution of mutants in various categories was almost the same, The Fox strain showed a much stronger mutation. This indicates the efficiency of the Fox strain, mainly due to the proper expression of full-length cDNA under the control of the strong CaMV promoter and nopaline synthesis (NOS) terminator.

In order to confirm the T ₂ generation mutant phenotype of the heritability of, grown a thin green mutant lines of 117, which appeared in the T ₁ generation (Table 2), was looking for a phenotype of T ₂ generation of mutation .

In order to monitor these phenotypes, 20 seedlings were grown each. Although it is difficult to establish a dominant dominance from 20 seedlings, 60 lines showed a light green phenotype (ie, the number of lines with a T ₂ phenotype rate of 50% or more in Table 2). 60). We also examined 40 morphological mutants and found 7 lines showing the phenotype of the original mutant. This is either due to T ₂ generation transgene inhibition is believed to have been identified as mutant incorrectly among T ₁ FOX plants. In particular, dwarf and leaf-shaped mutants are thought to have been caused by environmental stress after moving from the selection plate.

(5) Morphology caused by oncogenes Four oncogenes tms1, iaaM, rolB and tmr were mixed with the full-length cDNA library as internal standards. iaaM and tms1 encode tryptophan 2-monooxygenase from P. syringae and A. tumefaciens , respectively, and these genes are highly expressed to enhance the auxin response (Comai et al., 1982, J Bacteriol, 149, 40 -6). rolB is derived from Agrobacterium rhyzogenesis and is associated with susceptibility to auxin. tmr is an oncogene derived from Agrobacterium tumefaciens and functions in the biosynthesis of cytokinin. Each oncogene was mixed with full-length cDNA at a molar equivalent ratio of twice that of full-length cDNA.

  To monitor the expression and distribution of full-length cDNA, the appearance of oncogenes in the Arabidopsis FOX strain was examined. First, the morphology of the transformed plants was monitored for mutations caused by oncogenes. Only two had a morphological phenotype. Since the auxin response was enhanced in both tms1 and iaaM, apical dominance and a small phenotype were promoted in plants containing these genes (FIG. 2). These morphological differences are very clearly and easily distinguished from other morphological mutations. FIG. 2 (a) compares iaaM high expression plants and wild type (WT) plants grown during the same period. FIG. 2 (b) is a close-up photograph of a plant that highly expresses iaaM. FIG. 2 (c) is a plant that highly expresses tms1.

  High expression of tmr killed plant seedlings, but high expression of rolB did not show a clear phenotype. Among 15,547 Arabidopsis FOX strains, 13 high-expression mutants of tmr1 and iaaM were present. These apparent mutants were higher compared to the theoretical value (4 out of 15,000). This may be due to differences in the growth of transformed Agrobacterium in bacterial culture. One of the advantages of the oncogene is that the mutant is sterile, so other than rolB is not carried over to the next generation.

(6) Expression of FOX mutant by retransformation with full-length cDNA regenerated by PCR In order to further clarify and investigate the relationship between the introduced full-length cDNA and mutant phenotype, FOX mutants were selected from various forms of categories. F00721 and F00745 grew more slowly than the wild type (FIG. 3 (a): wild type, (b): F00721 and (c): F00745). F01049 had dark green leaves that were under-balanced growth (FIG. 3 (d)). F01310 had speckled leaves (FIG. 3 (e)). F02347 flowered at a very early stage of growth and had a very small morphology (FIG. 3 (f)). F02635 was a weak plant with a dominant apical bud (Fig. 3 (g)). After retransformation with full-length cDNA isolated by genomic PCR, all the original phenotypes were reproduced. The corresponding genes are summarized in Table 3. Very interestingly, it can be seen that an unknown gene is included in a gene that imparts a phenotype, and the phenotype is generated by overexpression of the gene.

(7) Comparison of wild-type plant and dwarfing gene transformed plant Wild-type (9 individuals) one month after transplantation had an average of 37.2 cm (standard deviation 3.7), and a trait that introduced the At1g66820 gene Converted plants (23 individuals) were 13.8 cm (standard deviation 8.2) (FIG. 4) and dwarfed to about one third of the wild type.

  The dwarf transformed plant of the present invention can be applied in the fields of agriculture and horticulture.

(a) shows a binary vector construct into which full-length cDNA has been introduced. (b) shows an electrophoresis image of the amplified cDNA. (c) shows the size distribution of RAFL cDNA. High expression phenotype or wild type phenotype of oncogene in Arabidopsis thaliana (a) shows the wild-type phenotype of Arabidopsis thaliana. (b)-(g) show the phenotypes of the strains related to Arabidopsis dwarfing. A comparison of the heights of the wild-type Arabidopsis thaliana and the transformed plant introduced with the dwarfing gene is shown.

Claims (6)

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2, or
(b) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having an activity of miniaturizing a plant can be expressed. A dwarf transformed plant. The gene is
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1, or
(d) a DNA encoding a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 and has an activity of dwarfing a plant body
The transformed plant of Claim 1 containing this.   The transformed plant according to claim 1, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.   A method for producing a dwarf transformed plant according to any one of claims 1 to 3, comprising regenerating a plant by introducing the gene defined in claim 1 or 2 into a plant tissue or cell. .   The method according to claim 4, wherein the gene is introduced using a recombinant vector containing the gene.   A transformed plant according to any one of claims 1 to 3, which comprises overexpressing the gene defined in claim 1 or 2 and inducing dwarfing.

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