JP2008271805A - Zim motif gene family yielding crop growth, flower blooming promotion and seed enlargement, and method for utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide genes promoting plant growth, and to provide a method for producing plants promoted in growth as a result of using the genes. <P>SOLUTION: Among Oryza sativa with the full-length cDNA transformed utilizing FOX Hunting system, strains (AB323 and AG163) where growth promotion is observed are expressed. The identical cDNA(AK067971) has been transduced in each of the AB323 and AG163 strains. AK067971 has ZIM motif, but there is no GATA-type Zinc Finger domain therein, and also has no domain having high homology with CCT motif. By further searching genes having characteristics similar to those of AK067971, a group of genes having characteristics similar to those of AK067971 has been found from Oryza sativa and a plurality of plants. Growth promotion in transformed Oryza sativa obtained using the group of genes has also been confirmed. Thus, AK067971 and its analogous genes can be utilized for plant growth promotion. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a rice ZIM motif gene family and use thereof.

  Plant genome research can enable short-term production of excellent trait varieties, which is difficult with conventional breeding techniques, by analyzing individual gene functions. Therefore, the search for genes that can impart useful traits to plants has been carried out at the world level.

  In 2004, the complete genome sequence of the rice genome was completed for rice, the main crop. Furthermore, the isolation of rice full-length cDNA has been promoted, and information on the isolated full-length cDNA clone has been made into a database and published. However, functional analysis of all genes has not been completed, and the discovery of new useful trait genes is expected.

  By the way, the ZIM protein derived from Arabidopsis thaliana is known as a protein belonging to the GATA transcription factor family (Non-patent Documents 1-3). It has been reported that when the ZIM gene was overexpressed in Arabidopsis thaliana using the CaMV 35S promoter, hypocotyl and petiole elongation was observed (Non-patent Document 3). The ZIM gene has, as structural features, a ZIM motif, a CCT motif, and a GATA type Zinc Finger domain (Non-patent Documents 1-3). The Zinc Finger domain is a structure often found in transcription factors, but the relationship between the ZIM motif and protein function is not known at all. In addition, in potato, tobacco (Non-patent Document 4), and poplar (Non-patent Document 5), it has been reported that a gene having a ZIM motif has been found, but the function of the gene is unknown.

Nishii et al. (2000) Biosci. Biotechnol. Biochem. 64: 1402-1409. Shikata et al. (2003) Biosci. Biotechnol. Biochem. 67: 2495-2497. Shikata et al. (2004) J. Exp. Bot. 55: 631-639. Katou et al. (2005) Plant Physiol. 139: 1914-1926. Major and Constabel (2006) New Phytol. 172: 617-635.

  The present invention has been made in view of the above situation, and the problem to be solved by the present invention is to provide a gene capable of imparting a useful trait to a plant and a method for using the gene, and more specifically, a crop. It is intended to provide a method for producing a gene that promotes the growth and flowering of plants, a gene that causes seed enlargement, and a plant in which the growth is promoted.

In order to solve the above problems, the present inventors have conducted extensive research. The present inventors have, to find a gene that provides a useful trait from the function unknown genes, using F ull-length cDNA O ver- e X pressor gene hunting system (FOX hunting system) (Ichikawa, T., Nakazawa, M., Kawashima, M., Iizumi, H., Kuroda, H., Kondou, Y., Tsuhara, Y., Suzuki, K., Ishikawa, A., Seki, M., Fujita, M., Motohashi, R., Nagata, N., Takagi, T., Shinozaki, K. and Matsui, M. (2006) The FOX hunting system: an alternative gain-of-function gene hunting technique.Plant J. 48: 974- 85., WO2003 / 018808). The FOX hunting system is a high-speed, genome-wide gene function search technique using full-length cDNA. From the rice transformed with rice full-length cDNA using the FOX hunting system, the present inventors observed significant growth promotion such as promotion of elongation of cocoons and roots, promotion of flower bud formation, and enlargement of seed endosperm. (AB323 and AG163) were found. Analysis of cDNA introduced into the AB323 and AG163 lines revealed that the same full-length cDNA (accession number: AK067971) was introduced into both AB323 and AG163. AK067971 cDNA encodes a ZIM motif protein consisting of 187 amino acids. ZIM motifs are Arabidopsis ZIM (Z inc-finger protein expressed in I nflorescence M eristem) first discovered the unknown function motif protein. The 309 amino acid Arabidopsis ZIM protein contains a GATA-type zinc finger domain and a CCT motif in addition to the ZIM motif, whereas the AK067971 gene product does not have a GATA-type zinc finger domain and does not contain a typical CCT motif sequence. . Therefore, the present inventors searched for a gene having the same characteristics as AK067971, further transformed rice with the gene, and evaluated its phenotype. Then, it became clear that the gene group which has the characteristic similar to AK067971 has the growth promotion effect of a plant like AK067971. Therefore, AK067971 and its related genes can be used to promote plant growth. That is, the present invention relates to a gene containing a ZIM motif and not including a GATA-type Zinc Finger domain and a typical CCT motif sequence, and uses thereof, and specifically provides the following inventions.

(1) Overexpressing a polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and a typical CCT motif and no GATA-type zinc finger domain in the plant. A method for promoting plant growth, characterized by
(2) The method according to (1) above, comprising the following steps (a) and (b): (a) a typical CCT motif and GATA type having an activity of promoting plant growth and including a ZIM motif in the amino acid sequence A step of introducing a polynucleotide encoding a protein not containing a Zinc Finger domain or a vector carrying the polynucleotide into a plant seed, fertilized egg, plant cell or callus (b) the seed, fertilized egg, plant cell, Or a process of regenerating a plant from callus,
(3) A method for producing a plant whose growth is promoted, comprising the following steps (a) to (c): (a) a typical CCT motif having an activity of promoting the growth of a plant and containing a ZIM motif in the amino acid sequence; A step of introducing a polynucleotide encoding a protein not containing a GATA-type zinc finger domain, or a vector carrying the polynucleotide into a plant seed, fertilized egg, plant cell or callus (b) the seed, fertilized egg, plant A step of regenerating the plant from cells or callus (c) a step of regenerating the plant from propagation material (other than seeds) derived from the plant,
(4) A method for producing a plant whose growth is promoted, comprising the following steps (a) to (c): (a) a typical CCT motif having an activity of promoting the growth of a plant and containing a ZIM motif in the amino acid sequence; A step of introducing a polynucleotide encoding a protein not containing a GATA-type zinc finger domain, or a vector carrying the polynucleotide into a plant seed, fertilized egg, plant cell or callus (b) the seed, fertilized egg, plant A step of producing a plant from cells or callus (c) a step of producing a plant overexpressing a gene having a ZIM motif from a seed having the polynucleotide obtained from the plant;
(5) A polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and not a typical CCT motif and a GATA type Zinc Finger domain is described below (a) to (d). The method according to any one of (1) to (4) above, wherein (a) any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 A polynucleotide comprising the coding region of the base sequence described in (b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 (C) a polynucleotide comprising a nucleotide sequence coding for SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 that is stringent to the complementary strand of the nucleotide sequence described in any one of Polynucleotide that hybridizes under conditions (d) SEQ ID NO: 2, 4, 6, 8, 10 , 12, 14, 16, 18, or 20 comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, or added to the amino acid sequence of any one of ,
(6) The growth of the plant is any one or more of plant height, root or cocoon elongation, seed endosperm hypertrophy, flowering / heading promotion, according to (1) to (5) above Method,
(7) The method according to any one of (1) to (6) above, wherein the plant is Gramineae, Brassicaceae, Eggplant, Cucurbitaceae, Convolvulaceae, Willowaceae,
(8) Isolation of the following (a) to (d) encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and no typical CCT motif and GATA type Zinc Finger domain Polynucleotide (a) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 comprising the coding region of the nucleotide sequence described in (b) sequence No .: 2, 4, 6, 8, 10, 12, 14, 16, 18, or a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence of (20) (c) SEQ ID NO: 1, A polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence according to any one of 3, 5, 7, 9, 11, 13, 15, 17, or 19 (d) SEQ ID NO: 2, 4, Amino acid according to any of 6, 8, 10, 12, 14, 16, 18, or 20 Column one or more amino acid substitutions, insertions, deletions, a polynucleotide comprising a nucleotide sequence encoding a protein consisting added in the amino acid sequence,
(9) A transformed plant cell that overexpresses the polynucleotide according to (8) above,
(10) A transformed plant body with enhanced growth, comprising the transformed cell according to (9) above,
(11) A transformed plant body whose growth is promoted, which is a descendant or clone of the transformed plant body according to (10),
(12) A propagation material of the transformed plant according to (10) or (11) above,
(13) Plant growth promoter containing the polynucleotide of any one of (a) to (d) below: (a) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, Or a polynucleotide comprising the coding region of the base sequence described in any one of (19) (b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 A polynucleotide comprising a base sequence encoding a protein comprising an amino acid sequence (c) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 complementary to the base sequence A polynucleotide that hybridizes to the strand under stringent conditions (d) one or more of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 A base sequence encoding a protein consisting of an amino acid sequence in which the amino acid is substituted, inserted, deleted, or added. Polynucleotide comprising,
(14) A plant growth promotion kit comprising any of the polynucleotides (a) to (d) below: (a) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, Or a polynucleotide comprising the coding region of the base sequence described in any one of (19) (b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 A polynucleotide comprising a base sequence encoding a protein comprising an amino acid sequence (c) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 complementary to the base sequence A polynucleotide that hybridizes to the strand under stringent conditions (d) one or more of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 A base that encodes a protein consisting of an amino acid sequence substituted, inserted, deleted, or added. Polynucleotide comprising a column,

  INDUSTRIAL APPLICABILITY According to the present invention, a novel gene having a plant growth promoting effect and a method for producing a transformed plant whose growth is promoted by the gene are provided. When the gene of the present invention is used in the agricultural field, for example, in the case of rice, high yield is expected due to the enlargement of endosperm. In addition, the growth of roots and cocoons, that is, an increase in biomass is expected. In addition, the effect of promoting flowering induction has been confirmed, and it is considered possible to produce fertilized crops. Furthermore, the gene of the present invention is considered to be usable not only for rice but also for other grasses (wheat, barley, corn, sugarcane, sorghum, etc.), and is expected to lead to the solution of food problems. If it is a field other than the field of agriculture, the gene production of the present invention can be used to increase the amount of useful substances produced by the plant factory. It is thought that the effect of absorption / removal (environmental restoration) of environmental pollution by heavy metals and harmful gases can be improved. Thus, the present invention is not only expected to make a high contribution to the agricultural field, but can also be widely used in other industries.

Detailed Description of the Invention
The present invention provides a method of promoting plant growth by overexpressing a polynucleotide encoding a protein having an activity of promoting plant growth in the plant. The present inventors have analyzed the full-length cDNA of a gene whose function is unknown in rice and found a gene for a protein having an activity of promoting plant growth.

  The polynucleotide used in the method of the present invention (hereinafter referred to as “the polynucleotide of the present invention”) encodes “a protein having an activity of promoting plant growth”, and the protein has a ZIM motif in the amino acid sequence. Contains, but does not contain a GATA-type Zinc Finger domain and a typical CCT motif sequence.

  The ZIM motif is a motif of unknown function found in the ZIM gene product (or ZIM protein) derived from Arabidopsis thaliana. The ZIM motif is often found in transcription factors containing GATA-type Zinc Finger domains and other motifs. The Arabidopsis ZIM gene product has a CCT motif and a GATA type Zinc Finger domain on the C-terminal side of the ZIM motif. The most highly conserved amino acid sequence in the ZIM motif is represented by TIFFXG (SEQ ID NO: 31) or TIFYXG (SEQ ID NO: 32). Information on the ZIM motif can be obtained from the domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) by accession number: pfam06200.

  The CCT motif is a common structure in plant proteins and is rich in basic amino acids. It consists of about 45 amino acids, and a hypothetical nuclear localization signal is thought to exist in the latter half of the CCT motif. The CCT motif of the Arabidopsis CONSTANS (CO) protein is known to be involved in nuclear localization but is being found to have additional functions. The CCT motif of the CO protein is homologous to the yeast HEME ACTIVATOR PROTEIN2 (HAP2: functions in combination with HAP3 and HAP5) that binds to the CCAAT box present in various promoters of eukaryotes. HAP3 and HAP5 Interact with. It is also known that the second half of the CCT motif is homologous to the NF-YA2 region of HAP2 that binds to DNA, that is, the CCAAT box (Wenkel et al. (2006) Plant Cell 18: 2971-2984.). Information on the CCT motif can be obtained by the accession number: pfam06203 in the above-mentioned domain database.

The “typical CCT motif” in the present invention can be represented by the following amino acid sequence.
RearvlRYreKrk-RkfeK-iRYasRKa-Ae-RpRvkGRFvkr (SEQ ID NO: 33)
In the above sequence, the amino acids shown in capital letters indicate that the protein having the CCT motif is highly conserved, and the lower case letters indicate a relatively highly conserved region. A place indicated by “-(hyphen)” indicates that the storage stability is low. That is, in the amino acid sequence set forth in SEQ ID NO: 33, amino acids at positions 1, 7-8, 11, 15, 19, 22-23, 26-27, 30, 33, 35, 38-40 are highly Amino acids at positions 2-6, 9-10, 12-13, 16-18, 21, 24-25, 28, 31, 34, 36-37, 41-43 are relatively highly conserved, The amino acids at positions 14, 20, 29 and 32 are poorly conserved.

The Zinc Finger domain is a structural domain formed by folding a polypeptide chain around a zinc atom, and is frequently found in DNA-binding proteins such as transcription factors. Several types of Zinc Finger domains are known. In addition to TFIIIA, GAL4, and GATA types, the existence of Dof and WRKY types as plant-specific types has been reported. The GATA type is further classified into subtypes, including the CX ₂ -CX ₁₇ -CX ₂ -C (SEQ ID NO: 34) motif and the CX ₂ -CX ₁₈ -CX ₂ -C (SEQ ID NO: 35) motif A type including a CX ₂ -CX ₁₉ -CX ₂ -C (SEQ ID NO: 36) motif and a type including a CX ₂ -CX ₂₀ -CX ₂ -C (SEQ ID NO: 37) motif are known. The GATA type Zinc Finger domain in the present invention may be any of the above, but is preferably a type containing a motif represented by CX ₂ -CX ₂₀ -CX ₂ -C.

  As a protein containing a ZIM motif, a ZIM protein derived from Arabidopsis thaliana is known. The ZIM protein has a CCT motif and a GATA type Zinc Finger domain in addition to the ZIM motif (FIG. 6A). It is clearly different from the encoded protein.

  Examples of the above-described sequences “including the ZIM motif in the amino acid sequence but not including the GATA-type Zinc Finger domain and the typical CCT motif sequence” are registered in a database (http: //www.ncbi.nlm.nih .gov / Structure / lexington / lexington.cgi? cmd = cdd & uid = 46091). As of April 5, 2007, 79 sequences have been registered.

  As long as the polynucleotide of this invention has the said characteristic, origin is not ask | required in particular, Preferably it is plant origin. As described in the Examples, the polynucleotide of the present invention is not only rice (Gramineae), but also Arabidopsis (Brassicaceae), potato (Solanum), Bensamiana tobacco (Solanum), cucumber (Cucurbitaceae), morning glory (Convolvulaceae) and tomatoes (Solanumaceae) are also present and are thought to be widely distributed in the plant kingdom. Accordingly, the plant from which the polynucleotide of the present invention is derived is not particularly limited, but is preferably derived from a monocotyledonous plant, more preferably from a grass family.

Specific examples of the polynucleotide of the present invention include a polynucleotide containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. it can. As shown in the Examples, the present inventors confirmed that plant growth is promoted by overexpression of the polynucleotide.
SEQ ID NO: 1 is published on the database as Accession Number: AK067971. The deduced amino acid sequence encoded by SEQ ID NO: 1 is shown in SEQ ID NO: 2. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 2 is shown in SEQ ID NO: 21.
SEQ ID NO: 3 is disclosed on the database as Accession Number: AK061602. The deduced amino acid sequence encoded by SEQ ID NO: 3 is shown in SEQ ID NO: 4. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 4 is shown in SEQ ID NO: 22.
SEQ ID NO: 5 is disclosed on the database as Accession Number: AK107854. The deduced amino acid sequence encoded by SEQ ID NO: 5 is shown in SEQ ID NO: 6. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 6 is shown in SEQ ID NO: 23.
SEQ ID NO: 7 is published on the database as Accession Number: AK070649. The deduced amino acid sequence encoded by SEQ ID NO: 7 is shown in SEQ ID NO: 8. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 4 is shown in SEQ ID NO: 24.
SEQ ID NO: 9 is published on the database as Accession Number: AK073589. The deduced amino acid sequence encoded by SEQ ID NO: 9 is shown in SEQ ID NO: 10. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 10 is shown in SEQ ID NO: 25.
SEQ ID NO: 11 is published on the database as Accession Number: AK108738. The deduced amino acid sequence encoded by SEQ ID NO: 11 is shown in SEQ ID NO: 12. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 12 is shown in SEQ ID NO: 26.
SEQ ID NO: 13 is published on the database as Accession Number: AK068566. The deduced amino acid sequence encoded by SEQ ID NO: 13 is shown in SEQ ID NO: 14. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 14 is shown in SEQ ID NO: 27.
SEQ ID NO: 15 is disclosed on the database as Accession Number: AK099557. The deduced amino acid sequence encoded by SEQ ID NO: 15 is shown in SEQ ID NO: 16. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 16 is shown in SEQ ID NO: 28.
SEQ ID NO: 17 is disclosed on the database as Accession Number: AK107750. The deduced amino acid sequence encoded by SEQ ID NO: 17 is shown in SEQ ID NO: 18. The sequence of the ZIM motif in the amino acid sequence described in SEQ ID NO: 18 is shown in SEQ ID NO: 29.
SEQ ID NO: 19 is disclosed on the database as Accession Number: AK069326. The deduced amino acid sequence encoded by SEQ ID NO: 19 is shown in SEQ ID NO: 20. The sequence of the ZIM motif in the amino acid sequence set forth in SEQ ID NO: 20 is shown in SEQ ID NO: 30.

  The polynucleotide described in the above SEQ ID NO can be prepared by a known technique. For example, total mRNA is prepared from a rice tissue extract, a primer is designed based on the base sequence described in the above SEQ ID NO, and a RACE method or the like is performed to obtain a full-length cDNA having the above SEQ ID NO. Alternatively, a cDNA library can be prepared from a rice tissue extract, a probe can be designed based on the base sequence described in the above SEQ ID NO, and obtained by a hybridization method. Further, it may be artificially synthesized based on the base sequence described in the above SEQ ID NO.

  Further, the method of the present invention provides a polynucleotide having a sequence similar to the base sequence described in the above SEQ ID No. and having a function equivalent to the base sequence described in the above SEQ ID No., that is, an activity for promoting plant growth. It can also be used. Therefore, the polynucleotide of the present invention includes a polynucleotide having a sequence similar to the base sequence described in the above SEQ ID NO and having an activity of promoting plant growth. Such a polynucleotide may be prepared from nature or artificially prepared as long as it has an activity of promoting plant growth. For example, orthologues, homologues of the above sequence numbers, or artificially mutated ones may be used. Examples of such polynucleotides include “stringent conditions to the complementary strand of the base sequence described in any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. Hybridizing polynucleotide ”and“ SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 with one or more amino acid substitutions or insertions, And a polynucleotide comprising a base sequence encoding a protein comprising a deleted or added amino acid sequence.

  Those skilled in the art can appropriately select the stringent hybridization conditions. For example, 25% formamide, 50% formamide under more severe conditions, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt's solution, hybridization solution containing 20 μg / ml denatured salmon sperm DNA at 42 ° C. After overnight prehybridization, add a labeled probe and incubate at 42 ° C. overnight. The cleaning solution and temperature conditions for the subsequent cleaning are about 1xSSC, 0.1% SDS, 37 ° C, more severe conditions are about 0.5xSSC, 0.1% SDS, 42 ° C, and more severe conditions are about 0.2xSSC. , 0.1% SDS, about 65 ° C. ”. Thus, isolation of a polynucleotide having high homology with the probe sequence can be expected as the conditions for washing for hybridization become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.

  A polypeptide encoded by a polynucleotide isolated using such a hybridization technique usually has high homology with the amino acid sequence shown in the above SEQ ID NO. High homology means at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95% or more, more preferably at least 97% or more (for example, 98% or more). To 99%) sequence homology. The identity of the amino acid sequence is determined by, for example, the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Can be determined by. Based on this algorithm, a program called BLASTX has been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

  In addition, the polynucleotide into which the mutation is introduced is subjected to site-specific or random mutation by subjecting the base sequence described in the above SEQ ID No. to gene modification methods well known to those skilled in the art such as PCR-mediated mutation introduction and cassette mutation. Can be prepared by introducing. Alternatively, it is also possible to synthesize a sequence in which a mutation is introduced into the base sequence described in the above SEQ ID No. using a commercially available nucleic acid synthesizer. The mutation may be introduced at any position as long as it has an activity of promoting the growth of the plant by overexpressing the polynucleotide in the plant. Preferably, the mutation is introduced at a position other than the ZIM motif, in which case the ZIM motif sequence remains intact in the polynucleotide.

  Confirmation of whether or not the polynucleotide prepared as described above has the activity of promoting the growth of the plant by over-expressing the plant in the plant is introduced and cultivated by introducing the prepared polynucleotide into the plant, In comparison, it may be evaluated whether plant height, root or cocoon elongation, seed enlargement, flowering induction, and heading promotion are observed. Specifically, it can be carried out by the method described in Example 2.

  In order to overexpress the polynucleotide of the present invention in a plant, the polynucleotide of the present invention is operably linked downstream of an appropriate promoter, and the polynucleotide of the present invention linked to the promoter is electroporated, particle What is necessary is just to introduce | transduce into a plant cell, a seed, a gamete, a fertilized egg, or a callus by well-known methods, such as a cancer method, a polyethyleneglycol method, an Agrobacterium infection method.

  Examples of the promoter include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the rice actin promoter, the elongation factor 1β promoter, the corn ubiquitin promoter, and the like. In addition, when it is desired to promote the growth of a specific organ, a promoter that can be expressed in a tissue-specific manner can be used. For example, if a promoter of a flower organ-specific gene is used, it is considered possible to concentrate the growth promoting effect of the polynucleotide of the present invention on high yield.

  When performing the Agrobacterium method, a binary vector or an intermediate vector can be used. Specific examples of binary vectors include pBI vectors (eg, pRiceFOX used in the Examples), pPZP systems (Hajdukiewicz, P., Svab, Z. and Maliga, P. (1994) The small, versatile pPZP family of Agrobacterium binary Plant molecular Biology 25 (6): 989-94.), pCAMBIA system (vector backbone: pPZP vector; URL: http://www.cambia.org/daisy/cambia/materials/vectors/585. html), pSMA system (Igasaki, T., Mohri, T., Ichikawa, H. and Shinohara, K. (2000) Plant Cell Reports 19: 448-453.), but is not limited thereto. The vector to be used may be equipped with a selection marker such as hygromycin resistance or kanamycin resistance.

  Since the plant into which the polynucleotide of the present invention is introduced is promoted by overexpression of the polynucleotide, the polynucleotide of the present invention can be used for the production of a plant whose growth is promoted. Growth promotion can be confirmed by traits such as plant height, root or pod elongation, seed enlargement, flower induction, and heading promotion. It is possible to mass-produce plants whose growth has been promoted by regenerating plants from propagation materials (for example, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) obtained from this introduction. . In addition, since it has been confirmed that this trait is stably transmitted to progeny, a plant whose growth is promoted can also be produced from seeds obtained by mating the current generation.

  The polynucleotide of the present invention can be packaged in an appropriate container together with instructions for use, and processed and distributed as a plant growth promoter or a growth promotion kit.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
[Example 1] Production of transgenic plants by rice FOX hunting system Binary vector for gene overexpression A variety of full-length cDNAs are connected downstream of the maize-derived ubiquitin 1 gene promoter (PUbi1) on pRiceFOX, and the plasmid mixture is Introduced into Agrobacterium, rice cDNA Agrobacterium library was prepared. The pRiceFOX vector has P35S-HPT (hygromycin resistance gene under the control of CaMV 35S promoter expression) as a selection marker. The structure of the pRiceFOX vector is shown in FIG. The library was transformed rapidly (WO 2005/092082, Toki, S., Hara, N., Ono, K., Onodera, H., Tagiri, A., Oka, S. and Tanaka, H. (2006 ) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J. 47: 969-976.) In the description, rice (transformation generation) regenerated from the transformed callus into which the gene was introduced was created.
Observation of the phenotype of the FOX rice line confirmed the appearance of lines (AB323 and AG163) whose growth was significantly promoted (FIG. 2). Analysis of the cDNA introduced into the AB323 and AG163 rice lines using genomic PCR and direct sequencing revealed that the same cDNA (accession number AK067971) was introduced into both lines. AK067971 encodes a protein consisting of 187 amino acids and has a ZIM motif. The base sequence of AK067971 is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the ORF is from position 108 to position 671.

[Example 2] Phenotypic analysis of AK067971 overexpressing rice line The characteristics of the progenies of AB323 and AG163 lines into which the overexpressed AK067971 cDNA was introduced were further analyzed. The phenotype in the AB323 line (T3) is shown in FIG. In the AB323 strain seedlings 12 days after sowing, the promotion of root elongation was observed. In seeds, endosperm hypertrophy was observed, and this phenotype was stably transmitted to progeny (FIG. 3, Table 1). In addition, the plant height, culm length, seed weight, and yield per individual were significantly higher than the control (FIG. 4).

(Table 1 Comparison of seed weight of AB323 line and control)

  In addition to the AB323 and AG163 lines, a plasmid (AK067971-FOX) in which AK067971 was individually introduced into the pRiceFOX vector was reintroduced into wild-type rice to further obtain the AK067971-FOX rice line. When the phenotypes of these novel AK067971-FOX rice lines were investigated, the same growth-promoting traits as the AB323 and AG163 lines were reproduced. Also, the heading date of the new line was found to be about 10 days earlier than that of the wild type (non-recombinant) as in the AB323 and AG163 lines (FIG. 5).

[Example 3] Phenotypic analysis of AK067971 gene homolog introduction line
The nucleotide sequence database was searched for the presence of a gene having a ZIM motif in addition to AK067971. As a result, it was found that ZIM motifs exist in a plurality of rice genes (Table 2). In addition, Arabidopsis thaliana, potato (Solanum tuberosum), Bensamiana tobacco (Nicotiana benthamiana), cucumber (Cucumis sativus), morning glory (Ipomoea nil), tomato (Lycopersicon esculentum), and Zenigoke (Marchantia polymorpha) Genes with a were found (Table 2). The gene group with the ZIM motif mainly has the ZIM motif, but does not have the GATA type Zinc Finger domain and does not have a region having high homology with the CCT motif, and the ZIM motif, CCT motif and GATA type Zinc It is roughly divided into two types having three structures of Finger domains. The AK067971 gene belongs to type 1 (FIG. 6A). When these ZIM motif gene family members were overexpressed in plants, the same effects as those observed in AK067971-FOX rice were expected.
9 rice homologs other than AK067971 that were confirmed to have the ZIM motif (AK107854, AK070649, AK061602, AK073589, AK068566, AK099557, AK108738, AK069326, AK107750) were compared with AK067971. As a result, homology at the amino acid level is AK107854: 45.3%, AK070649: 39.0%, AK061602: 44.0%, AK073589: 41.4%, AK068566: 20.5%, AK099557: 24.5%, AK108738: 26.7%, AK069326: 21.1% , AK107750: 27.2% (FIG. 6B). They were incorporated into pRiceFox vector in descending order of homology and transformed into rice. The T1 re-differentiated generation plant group into which the four homologs AK107854, AK070649, AK061602, and AK073589 were introduced showed values equivalent to or higher than those of the AK067971 introduced line in terms of plant height, culm length, and seed weight (FIG. 7). From these results, it has been clarified that overexpression of AK067971-related gene having ZIM motif gene also promotes plant growth.

(Table 2 Genes with ZIM motif)

It is a figure which shows the structure of the binary vector pRiceFOX for rice full length cDNA overexpression. It is the photograph which shows AB323 and AG163 strain | stump | stock which showed growth and flowering promotion by overexpression of AK067971 cDNA. (A): Growth status of AB323 line (T1 regeneration period) at 14 weeks after potting. Compared with the control, the plant height is higher and the heading is observed, and it can be seen that the mascot character is given. (B): A photograph showing the morphology of the AG163 line (T1) in the harvest period. Long cocoons and enlarged seed endosperm led to an increase in panicle weight. It is a photograph which shows the phenotype in AB323 system | strain (T3). (A): AB323 strain seedlings on the 12th day after sowing showed enhanced root elongation compared to the control. (B): Photograph showing endosperm hypertrophy in AB323 seeds. This phenotype was stably transmitted to the progeny. It is a graph which shows the growth promotion character in AB323 and AG163 strain | stump | stock. In all experiments, T2 progeny rice was used. ABOV1 and AG0V4 are transgenic controls into which an empty vector has been introduced. The numbers of individuals are AB323: n = 7, AG163: n = 4, AB0V1: n = 5, and AG0V4: n = 7, respectively. (A) A photograph showing the promotion of growth and heading of the AK067971 cDNA reintroduced individual (T1 generation) at 14 weeks after potting. Plants were grown under long day conditions (14L / 10D). (B) AK067971 is a graph showing the growth curve (plant height) of cDNA reintroduced individuals. (C) A graph showing the average heading date of reintroduced individuals. The average heading date for the control (n = 13) is 100.7 days, and the average heading date for the reintroduced line (n = 24) is 90.1 days. (A) It is a figure which shows typically the structure of the gene group which has a ZIM motif. It has a ZIM motif but no GATA type Zinc Finger domain and has three structures of ZIM motif, CCT motif and GATA type Zinc Finger domain. Broadly divided into types. (B) A phylogenetic tree of the type 1 ZIM gene family showing high homology to the AK067971 amino acid sequence. Gene products with accession numbers starting with AK are from rice. It is a graph which shows the phenotype comparison of the rice (T1 generation) which overexpresses cDNA of a type 1 ZIM gene family. Each rice was cultivated under long day conditions (14L / 10D).

Claims (14)

A polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and not a typical CCT motif and a GATA-type zinc finger domain is overexpressed in the plant. , A method of promoting plant growth. The method of claim 1, comprising the following steps (a) and (b).
(A) a polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and not including a typical CCT motif and a GATA-type Zinc Finger domain, or a vector retaining the polynucleotide (B) Step of regenerating a plant body from the seed, fertilized egg, plant cell, or callus A method for producing a plant whose growth is promoted, comprising the following steps (a) to (c):
(A) a polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and not including a typical CCT motif and a GATA-type Zinc Finger domain, or a vector retaining the polynucleotide (B) introducing into plant seed, fertilized egg, plant cell or callus (b) regenerating plant body from the seed, fertilized egg, plant cell, or callus (c) propagation material derived from the plant body (other than seed) The process of regenerating the plant from A method for producing a plant whose growth is promoted, comprising the following steps (a) to (c):
(A) a polynucleotide encoding a protein having an activity of promoting plant growth and containing a ZIM motif in the amino acid sequence and not including a typical CCT motif and a GATA-type Zinc Finger domain, or a vector retaining the polynucleotide (B) introducing into a plant seed, fertilized egg, plant cell or callus (b) producing a plant from the seed, fertilized egg, plant cell or callus (c) the polynucleotide obtained from the plant Of producing a plant overexpressing a gene having a ZIM motif from seeds having A polynucleotide encoding a protein having an activity to promote plant growth and containing a ZIM motif in the amino acid sequence and not a typical CCT motif and a GATA-type zinc finger domain is any of the following (a) to (d): The method according to claim 1, wherein:
(A) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 containing the coding region of the base sequence described in (b) SEQ ID NO: 2, A polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence of any one of 4, 6, 8, 10, 12, 14, 16, 18, or 20 (c) SEQ ID NO: 1, 3, 5, A polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence according to any one of 7, 9, 11, 13, 15, 17, or 19 (d) SEQ ID NOs: 2, 4, 6, 8, Poly comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, or added to the amino acid sequence of any one of 10, 12, 14, 16, 18, or 20 nucleotide The method according to claim 1, wherein the promotion of plant growth is one or more of plant height, root or cocoon elongation, seed endosperm hypertrophy, and flowering / heading promotion. The method according to any one of claims 1 to 6, wherein the plant is Gramineae, Brassicaceae, Eggplant, Cucurbitaceae, Convolvulaceae, or Willowaceae. The isolated poly (1) to (d) below, which has an activity of promoting plant growth and encodes a protein containing a ZIM motif in the amino acid sequence and not a typical CCT motif and a GATA-type zinc finger domain: nucleotide.
(A) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 containing the coding region of the base sequence described in (b) SEQ ID NO: 2, A polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence of any one of 4, 6, 8, 10, 12, 14, 16, 18, or 20 (c) SEQ ID NO: 1, 3, 5, A polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence according to any one of 7, 9, 11, 13, 15, 17, or 19 (d) SEQ ID NOs: 2, 4, 6, 8, Poly comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, or added to the amino acid sequence of any one of 10, 12, 14, 16, 18, or 20 nucleotide A transformed plant cell overexpressing the polynucleotide of claim 8. A transformed plant body comprising the transformed cell according to claim 9, the growth of which has been promoted. A transformed plant body whose growth is promoted, which is a descendant or clone of the transformed plant body according to claim 10. The propagation material of the transformed plant body of Claim 10 or 11. A plant growth promoter comprising the polynucleotide of any one of (a) to (d) below:
(A) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 containing the coding region of the base sequence described in (b) SEQ ID NO: 2, A polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence of any one of 4, 6, 8, 10, 12, 14, 16, 18, or 20 (c) SEQ ID NO: 1, 3, 5, A polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence according to any one of 7, 9, 11, 13, 15, 17, or 19 (d) SEQ ID NOs: 2, 4, 6, 8, Poly comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, or added to the amino acid sequence of any one of 10, 12, 14, 16, 18, or 20 nucleotide A kit for promoting plant growth, comprising a polynucleotide according to any one of (a) to (d) below.
(A) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 containing the coding region of the base sequence described in (b) SEQ ID NO: 2, A polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence of any one of 4, 6, 8, 10, 12, 14, 16, 18, or 20 (c) SEQ ID NO: 1, 3, 5, A polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence according to any one of 7, 9, 11, 13, 15, 17, or 19 (d) SEQ ID NOs: 2, 4, 6, 8, Poly comprising a nucleotide sequence encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, or added to the amino acid sequence of any one of 10, 12, 14, 16, 18, or 20 nucleotide