JP2008228713A - Nucleic acid for controlling formation of shape of plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for controlling shape formation in plants such as trees. <P>SOLUTION: There are provided: a nucleic acid selected from a group consisting of DNA and RNA of(a) a DNA composed of a specific base sequence derived from a specific plant of the genus Eucalyptus, (b) a DNA composed of a base sequence having at least 80% identity with the base sequence, (c) a DNA which is complementary to the DNA (a), (d) a DNA which is complementary to the DNA (b), (e) an RNA encoding the DNA (a), (f) an RNA encoding the DNA (b), (g) an RNA encoding the DNA (c) and (h) an RNA encoding the DNA (d), used for control of shape formation of plants; or a nucleic acid combination composed of a plurality of the nucleic acids; a microarray containing the nucleic acid combination; a specific base sequence derived from the plant of the genus Eucalyptus; or a nucleic acid which is a siRNA for use of shape formation of a plant, encoding a double strand DNA containing a base sequence having at least 80% identity with the sequence and a base sequence which is complementary to the base sequence; or its nucleic acid combination. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to nucleic acids and methods for controlling the morphogenesis of plants, particularly tree plants.

  The world's timber consumption is increasing every year, and by use, the consumption of wood-burning wood accounts for a majority, and it is still increasing in developing regions. Consumption of industrial materials such as lumber and wood chips for papermaking has also been increasing in the developing countries, although the consumption in developed regions has slightly decreased. In advanced areas, with the development of civilization, forests have been greatly reduced by diverting forests to farmland and using them as materials, but recently they have increased slightly due to tree planting activities. However, in the developing regions, the demand for living fuel and farmland has increased in recent years due to commercial logging in developed countries, and as a result, the area of forests in the world has been rapidly decreasing. On the other hand, soil degradation caused by salt damage, desertification, etc. is rapidly progressing, which is a big problem worldwide.

  If genetically modified trees can be obtained, individuals with excellent growth potential, individuals with modified xylem components, or land where plants cannot grow due to drought or salt damage due to improved stress adaptability There is a possibility of creating individuals.

  In recent years, as a gene expression control mechanism, unlike the transcriptional control of a gene, a control mechanism by post-transcriptional translational repression has been clarified, suggesting that the essence is a low molecular weight RNA. This is an important mechanism for determining the final gene expression effect by regulating the step of the transcript being converted to protein, following the essential and fundamental main functions such as gene transcription control. (Non-patent Documents 1 and 2).

Experimental Medicine, "RNAi Science", Vol. 22, No. 4, 2004, Yodosha Chisato Ushida, Protein Nucleic Acid Enzyme, Vol.46 No.10, 1381-1386, Kyoritsu Shuppan

  An object of the present invention is to provide a means for controlling various morphogenesis in plants, particularly trees.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a very large number of microRNAs are supplemented in tissues of certain tree plants, and they are expressed, and these microRNAs are expressed. Concludes that this is related to the morphogenesis control mechanism, translation control mechanism, etc. of the tree plant, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A nucleic acid selected from the group consisting of DNA and RNA of the following (a) to (h) or a nucleic acid combination consisting of a plurality of nucleic acids for use in controlling plant morphogenesis.
(a) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 751
(b) DNA comprising a base sequence having at least 80% identity with any one of SEQ ID NOs: 1 to 751
(c) DNA complementary to the DNA of (a) above
(d) DNA complementary to the DNA of (b) above
(e) RNA encoding the DNA of (a) above
(f) RNA encoding the DNA of (b) above
(g) RNA encoding the DNA of (c) above
(h) RNA encoding the DNA of (d) above

(2) encodes a double-stranded DNA comprising any one of SEQ ID NOs: 1 to 751 or a base sequence having at least 80% identity with the sequence and a base sequence complementary to the base sequence A nucleic acid that is an siRNA for use in controlling plant morphogenesis, or a nucleic acid combination thereof.
(3) The nucleic acid or nucleic acid combination according to (1) or (2) above, wherein the plant is a tree.
(4) The nucleic acid or nucleic acid combination according to (3) above, wherein the tree is a Eucalyptus plant.
(5) The nucleic acid or the nucleic acid combination thereof according to (2) above, wherein the siRNA further comprises an overhang sequence at the 3 ′ end of the sense strand and the antisense strand.

(6) The nucleic acid or the nucleic acid combination thereof according to (2) above, wherein the siRNA further comprises a loop sequence between the sense strand and the antisense strand.
(7) A vector comprising a DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 751, or a nucleotide sequence having at least 80% identity with the sequence.
(8) A transformed microorganism or a transformed plant cell comprising the vector according to (7) above.
(9) A transformed plant comprising the transformed plant cell according to (8) above.
(10) The transformed plant according to (9) above, wherein the plant is a tree such as a Eucalyptus plant.

(11) The nucleic acid or the nucleic acid combination thereof according to any one of (1) to (6) above, the vector according to (7) above, or the transformed plant cell or transformed microorganism according to (8) above A method for controlling morphogenesis of a plant, characterized in that
(12) The method according to (11) above, which is control of organ morphogenesis.
(13) A microarray comprising a combination of nucleic acids selected from the group consisting of the nucleic acids (a) to (h) described in (1) above.
(14) The microarray according to (13) above, wherein the nucleic acid is RNA.
(15) The microarray according to (13) above, wherein the nucleic acid is DNA.
(16) The microarray according to any one of (13) to (15), wherein the microarray is for screening a substance that controls plant morphogenesis.

(16) The microarray according to any one of (13) to (15) above, which is for identifying a gene involved in plant morphogenesis, a transcription product or a translation product thereof.
(17) The microarray according to any one of (13) to (15) above, which is for identifying a gene involved in plant morphogenesis, a transcription product or a translation product thereof.
(18) A seed derived from the transformed plant according to (9) or (10) above.
(19) A progeny of the transformed plant according to (9) or (10) above.

  The nucleic acids according to the invention make it possible to control the morphogenesis of plants, preferably trees.

In the first aspect, the present invention provides the following DNAs and RNAs (a) to (h) for use in controlling plant morphogenesis:
(a) DNA comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 751;
(b) a DNA comprising a base sequence having at least 80% identity with any one of SEQ ID NOs: 1 to 751;
(c) DNA complementary to the DNA of (a) above;
(d) DNA complementary to the DNA of (b) above;
(e) an RNA encoding the DNA of (a) above;
(f) RNA encoding the DNA of (b) above;
(g) an RNA encoding the DNA of (c) above; and
(h) RNA encoding the DNA of (d) above
A nucleic acid selected from the group consisting of: or a nucleic acid combination consisting of a plurality of nucleic acids thereof.

  As used herein, “control of morphogenesis” refers to controlling organ formation in plants, and “control” refers to promotion or suppression. Such control functions include, for example, hypertrophic growth in plant vascular tissue, function of generating root hair in plant root organs, function of generating roots from stem tissue in cuttings, etc., in plant flower organs, This includes functions such as converting flower cover parts into reproductive organs.

  As used herein, “RNA encoding DNA” means that the RNA has a base sequence in which the base T in the base sequence of the DNA is replaced with U.

  The nucleic acid combination of the present invention refers to any combination of two or more nucleic acids selected from the DNAs and RNAs of (a) to (h) above, for example, selected from the DNAs of (a) to (d) above A combination of two or more nucleic acids, a combination of two or more nucleic acids selected from the RNAs of (e) to (h) above, (a), (b), (c), (d), (e) A combination of two or more selected from the DNAs or RNAs of (f), (g) or (h), a combination of two or more selected from the DNAs of (a) and (c) above, (b) and ( a combination of two or more selected from the DNA of d), a combination of two or more selected from the RNAs of (e) and (g) above, or a combination of two or more selected from the RNAs of (f) and (h) above And so on.

  Plants whose morphogenesis is controlled in the present invention include dicotyledonous and monocotyledonous angiosperms, gymnosperms, etc., preferably trees such as eucalyptus plants, poplar, pine, red oak, cedar, hinoki. More preferably, Eucalyptus globulus plants such as bamboo, yew, etc. Eucalyptus plants are not particularly limited. For example, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus camaludulensis, Eucalyptus camaludulensis, Eucalyptus nitens, Eucalyptus gunni, Eucalyptus radiata, Eucalyptus amplifolia, Eucalyptus archeri, Eucalyptus bax, Eucalyptus bax (Eucalyptus bicostata), Eucalyptus blakelyi, Eucalyptus botryoides, Eucalyptus bridgesiana, and the like.

  The microRNA corresponding to the DNA having the nucleotide sequence of SEQ ID NOs: 1 to 751 of the present invention can be sequenced and prepared from the tissues of Eucalyptus plants. As an experimental technique for that purpose, for example, a technique disclosed in Sambrook et al., Molecular Cloning A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press can be used. Specifically, for example, it can be obtained as follows. Low molecular RNA is separated from total RNA extracted and purified from tissue by acrylamide gel electrophoresis, etc., and once converted to cDNA, it is made into a library. Library creation by MPSS method (Brenner, S. et al., Nature Biotechnol. 18: 630-634 (2000)), 454 sequencing (Margulies, M. et al., Nature 437: 376-380 (2005)), etc. The sequence of the small RNA can be determined. The low molecular weight RNA fraction contains known RNAs such as ribosomal RNA and transfer RNA, and these sequences are excluded using known sequence information.

  The nucleic acid of the present invention includes a nucleotide sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identity with any one of SEQ ID NOs: 1 to 751. And its complementary DNA, as well as RNA encoding the DNA and complementary DNA.

  As used herein, the term “identity” refers to the percentage of the same number of bases relative to the total number of bases when the two sequences are aligned (ie, aligned). Sequence identity can be determined by accessing a sequence data bank such as NCBI (USA) and using a sequence homology search system using known algorithms such as BLAST and FASTA. FASTA searches for consecutively matching sequence fragments at high speed, performs local alignment focusing on those fragments with high similarity, and finally combines them after considering gaps On the other hand, BLAST divides the sequence into fixed-length fragments (words), searches for similar fragments in units of words, and stretches them in both directions until the degree of similarity is maximized. This is known as a method of performing local alignment and finally combining these to perform final alignment.

  Nucleotide sequence variations include variations based on individual differences due to differences in plant families, genera, species, varieties, etc., mutations such as mismatches, and artificial variations. Such a mutation is a mutation from any one of SEQ ID NOs: 1 to 751, and substitution, deletion or addition of 1 or 2 or more, preferably 1, 2, 3 or 4 bases is performed. Including. These mutations are within the scope of the invention as long as they confer activity that controls plant morphogenesis. Nucleic acids of the present invention, including variants, can be synthesized using an automated DNA / RNA synthesizer.

The nucleic acid shown in the above (a) to (h) of the present invention is, for example, (1) for identifying a gene involved in plant morphogenesis, its transcription product or its translation product, or (2) plant It can be used to screen for substances that control the morphogenesis of (3) or to directly or indirectly control the morphogenesis of plants. For the identification or screening of (1) and (2), a microarray in which a plurality of types of nucleic acids selected from the nucleic acids (a) to (h) are immobilized on a carrier can be used. On the other hand, the control of morphogenesis in (3) can be performed by RNA interference (RNAi) using a nucleic acid selected from the nucleic acids (a) to (h) in vivo. Here, RNAi refers to a phenomenon in which expression of a gene having a homologous sequence is suppressed by introduction of double-stranded RNA (dsRNA) into a cell.
The use of the nucleic acid of the present invention will be described below.

<Control of plant morphogenesis>
According to the second aspect of the present invention, the base sequence of any one of SEQ ID NOs: 1 to 751, or a base sequence having at least 80% identity with the sequence, and a base sequence complementary to the base sequence, SiRNA encoding a double-stranded DNA containing or a combination thereof can be used for the control of plant morphogenesis.

  As described above, the RNA strand of the siRNA of the present invention is at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identical to any one of SEQ ID NOs: 1 to 751. RNA encoding a nucleotide sequence having sex can be included. Such RNA strand sequence differences are based on variations such as variations based on individual differences due to differences in plant families, genera, species, varieties, etc., mutations such as mismatches, and artificial variations. The mutation includes 1 or 2 or more, preferably 1, 2, 3 or 4 base substitutions, deletions or additions in each sequence, cleaves the target mRNA, and inhibits or suppresses the production of the translation product. Any variation is possible as long as it is a sequence variation that can be made.

  The siRNA of the present invention is a double-stranded RNA comprising a sense strand sequence and an antisense strand sequence consisting of 18 to 25 bases, preferably 19 to 24 bases, more preferably 20 to 23 bases, and cleaves the target mRNA. Thus, it is possible to control the expression of the gene and thus the translation into a protein. Here, the sense strand sequence refers to a sequence that is substantially the same as the base sequence of the target mRNA, and the antisense strand sequence refers to a sequence that is complementary to the sense strand sequence.

  The siRNA of the present invention, in a preferred embodiment, includes an overhang (ie, a protruding end) at each 3 'end of the sense and antisense strands. The 3 'overhang can comprise 1 to 5, preferably 1 to 4, more preferably 1 to 3 bases, preferably U or poly U.

  The siRNA of the present invention may also have an unpaired single-stranded loop sequence interposed between the sense strand sequence and the antisense strand sequence. Intron sequences are often used as loop sequences. For example, the loop sequence contained in siRNA expression vector: pHANNIBAL shown by Wesley S. et al. (Plant Journal 27: 581-590 (2001)) is a typical example. However, it is not limited to these. Alternatively, two RNA strands in which the same base as the above overhang is linked to the 3 ′ end of each strand may be generated, and a single-stranded loop sequence may be similarly interposed between the sense strand and the antisense strand. . The hairpin dsRNA thus obtained can be processed in vivo by endogenous dicer to form the siRNA of the present invention.

The siRNA of the present invention can be directly introduced into a plant body, plant tissue or plant cell. Plants include angiosperms and gymnosperms, preferably trees such as Eucalyptus plants, poplars,
Pine, red oak, cedar, cypress, bamboo, yew and the like, more preferably Eucalyptus plants. Plant tissues include meristems such as stem and root growth points, formation layers, organs such as roots, stems, flowers, leaves, buds, hypocotyls, seeds, tubers, cuttings, pollen and the like.

  Examples of the introduction method include a particle gun method, a liposome method, an electroporation method, and the like. The particle gun method is a method in which a nucleic acid is coated on metal particles such as gold and the nucleic acid is driven into a cell like a shot gun. The liposome method is a method in which a nucleic acid is encapsulated in a liposome made of a phospholipid bilayer membrane similar to a cell membrane, and the nucleic acid is introduced into the cell through membrane fusion with the cell membrane and endocytosis. For this purpose, cationic liposomes are preferred. The electroporation method is a method in which a nucleic acid is introduced into a cell by applying an electric pulse to the cell to temporarily give fluctuation to the lipid bilayer.

  Another example of the method for introducing siRNA of the present invention is a plant body, plant tissue or plant in which a double-stranded DNA encoding the above siRNA (preferably siRNA containing a 3 ′ overhang) or hairpin dsRNA is incorporated into a vector. It is a method of introducing into cells. Such vectors are also encompassed by the present invention.

  Specifically, the nucleic acid of the present invention, that is, a vector containing a DNA comprising any one of SEQ ID NOS: 1 to 751 or a DNA comprising at least 80% identity with the sequence is introduced into a plant cell, and obtained. By regenerating a plant from the transformed plant cells thus obtained, a transformed plant with controlled organ morphogenesis can be obtained.

  A vector can be obtained by binding or inserting a double-stranded DNA comprising the above DNA and its complementary DNA into a predetermined site of the vector. The type of the vector is not particularly limited, and a vector that can replicate autonomously or can homologously recombine into a chromosome can be used. Examples of such vectors include plasmids, phages, viruses, cosmids and the like. The vector can appropriately include a selection marker, a replication origin, a terminator, a polylinker, a promoter, an enhancer, a ribosome binding site, and the like. Commercially available vectors and vectors described in literatures can be used as the vector. Examples of the promoter include a CaMV 35S promoter, a tissue-specific or time-specific promoter derived from a plant having inducibility by an external factor, and the like. Examples of selectable markers include drug resistance genes such as the kanamycin resistance gene, neomycin phosphotransferase II (NPTII), dihydrofolate reductase, and the like. Specifically, vectors that can be amplified by E. coli (pUC derivatives, etc.), shuttle vectors that can be amplified by both E. coli and Agrobacterium (pBI101 (Clontech), etc.), plant viruses (for example, cauliflower mosaic virus (CaMV)) Etc.) and can be selected depending on the host cell.

  For example, in the method using Agrobacterium, a T-DNA region which is a part of plasmid DNA inherent to Agrobacterium is excised, and between the RB (right border) and LB (left border), A step of incorporating a foreign nucleic acid, a step of inserting the obtained DNA fragment into a vector for E. coli (for example, pBR system, pBI system, pUC system, pBluescript system, etc.), a step of transforming E. coli with the obtained vector Joining the transformed Escherichia coli to Agrobacterium and transferring the vector to Agrobacterium, and infecting the plant cell with Agrobacterium having the foreign nucleic acid to incorporate the foreign nucleic acid into the cell genome.

  A transformed microorganism or a transformed plant cell into which the vector is introduced is also encompassed in the present invention. Here, the “transformed microorganism” is a microorganism obtained by introducing the vector into a microorganism as a host cell. Examples of the host microorganism include bacteria such as Agrobacterium (for example, Agrobacterium tumefaciens, Agrobacterium rhizogenes, etc.) and the like.

  In the present invention, the transformed plant cell is a transformed cell obtained by introducing the vector into a plant-derived cell as a host cell. Examples of plant tissues include meristems such as stem and root growth points, formation layers, organs such as roots, stems, flowers, leaves, buds, hypocotyls, seeds, tubers, cuttings, pollen, etc. Preferred tissues are propagation media such as seeds, tubers, cuttings, etc.

  As a method for introducing the vector into a host microorganism, plant cell, or plant tissue, a conventional method in this technical field, for example, a protoplast method (electroporation method, microinjection method, polyethylene glycol method, etc.), particle gun method, Alternatively, a plant by T-DNA using the above-mentioned genus Agrobacterium (for example, Agrobacterium tumefaciens, Agrobacterium rhizogenes) as a transforming factor A cell transformation method or the like can be used.

  The protoplast method, particle gun method, and specific methods using Agrobacterium as a transforming factor can be carried out based on, for example, the Plant Metabolic Engineering Handbook (NTS Corporation). .

  Alternatively, in the present invention, a transformed plant cell can be obtained by using a plant virus (cauliflower mosaic virus or the like) as a vector. Specifically, first, a plant virus genome is inserted into a vector derived from E. coli to prepare a recombinant, and then the DNA to be introduced is inserted into the plant virus genome. These DNAs can be introduced by excising the plant virus genome thus prepared from the recombinant with a restriction enzyme and inoculating it into plant cells. For details of this method, reference can be made to the method of Hohn et al. (Molecular Biology of Plant Tumors (Acade Micro c Press, New York) 1982, pp549), US Pat. No. 4,407,956, and the like.

  The present inventors have found that the nucleic acid of the present invention, ie the siRNA described above, enables translational control of plants, preferably trees, for example Eucalyptus plants. Accordingly, the present invention relates to the control of gene expression, including the use of the above-described nucleic acid of the present invention or a combination thereof, the vector of the present invention, or the transformed plant cell or the transformed microorganism of the present invention, thereby a plant. A method of controlling morphogenesis, preferably organogenesis, is provided.

Control of gene expression includes promotion of gene expression and suppression of gene expression, and control of organ morphogenesis includes the promotion and suppression of various organ formations. Both are included. That is, organ morphogenesis can be suppressed by promoting the expression of the gene comprising the RNA fragment of the present invention, and organ morphogenesis can be promoted by suppressing the expression of the gene comprising the RNA fragment of the present invention. it can.
The present invention further provides a transformed plant comprising the transformed plant cell.

  The transformed plant cell is preferably a tree cell, more preferably a transformed Eucalyptus plant cell, and the transformed plant is preferably a transformed tree, more preferably a transformed Eucalyptus plant.

  In the present invention, the transformed plant is not particularly limited as long as it is a plant having the transformed plant cell, and includes, for example, a transformed plant regenerated from the transformed cell. For the method of regenerating plant bodies from transformed plant cells, the method of Toi et al. (Japanese Patent Application No. 11-127025) can be referred to. In addition, the transformed plant of the present invention includes seeds obtained from the transformed plants and progenies that are plant bodies obtained from the seeds.

  As a method for obtaining seeds from the transformed plant of the present invention, for example, the transformed plant is rooted in an appropriate medium, and the rooted body is transplanted to a pot containing water-containing soil. The seeds are obtained by growing them under appropriate cultivation conditions and finally forming seeds. In addition, as a method for obtaining a plant from seeds, for example, seeds derived from the transformed plant obtained as described above are sown in water-containing soil and grown under appropriate cultivation conditions. Can be obtained.

<Identification of genes involved in plant morphogenesis and screening for substances that control plant morphogenesis>
The present invention further provides a microarray comprising a combination of nucleic acids selected from the nucleic acids (a) to (h) of the present invention.

  In this case, the nucleic acids of the above (a) to (h) are used as probes for identifying or screening a specific gene or the like or a specific regulatory substance (for example, agonist, antagonist, promoter, suppressor, etc.) It is possible to identify or determine a target gene or the like or a regulatory substance by detecting the hybridization.

Hybridization and washing conditions vary depending on the purpose. For example, conditions described in the manual of mirVana ^TM MiRNABioarray provided by Applied Biosystems (formerly Ambion), supervised by Masaaki Muramatsu and Hiroyuki Nami, DNA microarray and the latest PCR method The conditions described in Shujunsha (2000) are applicable. For example, hybridization is performed at room temperature (about 25 ° C.) to 65 ° C. in a solution containing 1 to 6 × SSC, and optionally 5 × Denhard's solution and 1 mg / ml denatured salmon sperm DNA. It is carried out at 55-65 ° C. in a solution containing 2 × SSC, 0.1-0.2% SDS.

  The microarray of the present invention can be used for identifying a gene involved in plant morphogenesis, a transcription product or a translation product thereof, or screening a substance that controls plant morphogenesis.

  Among the nucleic acids of (a) to (h), more preferred nucleic acids as probes are the DNAs of (a) to (d), for example, the DNA of (c) or (d), and (e) to (d). h) RNA, for example, the RNA of (g) or (h) above. The number of probes bound on the surface of the array is not particularly limited as long as it is 2 or more, but preferably 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 Or more, 500 or more, 550 or more, 600 or more, 650 or more, 700 or more, or 750 or more.

  The microarray substrate is not particularly limited as long as it can solidify nucleic acid, and examples thereof include glass, silicon, and polymer. The surface of the substrate may be subjected to a surface treatment such as coating with a poly L lysine coat or introduction of a functional group such as an amino group or a carboxyl group, if necessary.

  The solid-phase immobilization method is not particularly limited as long as it is a conventional method, and includes a method of synthesizing a polynucleotide on a substrate, a spotting of a polynucleotide with a spotter or the like on the substrate, and a method of spraying a polynucleotide with an inkjet or the like. The basic method for this is the Affymetrix method, that is, a method of synthesizing a polynucleotide of about 19 to 25 mer on a substrate, or the Stanford method, that is, a spotter having a size of several tens to several hundreds of μm. The method of spotting with etc. is known.

  Samples can be synthesized from total RNA extracted from tissues or cells at various stages of growth from plant development to plant body, and can be used, or further synthesized from cDNA by in vitro transcription. However, this may be used. As the label, fluorescent dyes such as Cy3 (green) and Cy5 (red) are usually used.

  By using the microarray of the present invention, a specific probe can be used in various stages of plant growth (eg, flower bud formation, rooting, etc.) or after an event such as drug treatment or cutting. By identifying a hybridized gene, it is possible to estimate the function of a specific gene at a specific stage or a specific event, or the relationship with plant morphogenesis.

  Based on knowledge obtained from microarray analysis, it becomes possible to control plant morphogenesis, for example, organogenesis, at the molecular level, control rooting and flower buds, promote growth, increase bulk, stress tolerance Giving, enlargement growth in plant vascular tissues, generating root hairs in plant root organs, generating roots from stem tissues in cuttings, converting flower coat parts into reproductive organs in plant flower organs, etc. Can be made possible. Here, control refers to promotion or suppression.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

  The DNA fragment of the present invention was produced and used as follows. The experimental procedure was carried out based on experimental documents known in the art such as “Molecular Cloning A Laboratory Manual” (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press) unless otherwise specified.

(Example 1) Acquisition of low molecular RNA from Eucalyptus (1) Extraction of total RNA Liquid 10-year Eucalyptus grandis growing in Oji Paper Co., Ltd. Forest Resource Research Institute (Kameyama, Mie Prefecture) After pulverization in nitrogen, RNA was extracted using Concert Plant RNA Regent (Invitrogen) to obtain 1 mg of total RNA.

(2) Purification of low-molecular RNA Ethanol precipitation and drying were carried out using the total amount of total RNA described above, and dissolved in 100 μL of DEPC-treated water. OD measurement was carried out using a part and the concentration was calculated, then 100 μg of total RNA was run on a 15% denaturing polyacrylamide gel, and a small RNA fraction corresponding to 18 to 26 bases was separated and collected. At this time, 18mer and 26mer synthetic RNAs were used as markers. Moreover, small RNA Gel Extraction Kit (TaKaRa Bio) was used for collection of the small RNA fraction from the acrylamide gel. Finally, elution was performed with 40 μL of DEPC-treated water, and then a purity check and quantification were performed with an Agilent 2100 Bioanalyzer using a part of the sample.

(3) Preparation of cDNA library derived from low molecular RNA and determination of nucleotide sequence Megaclone microbeads were prepared using the purified small RNA fraction. Each operation is basically performed according to literature methods (Brenner et al. Proc Natl Acad Sci USA 97 (4): 1665-1670; Brenner et al. Nat. Biotechnol 18 (6): 630-634; Ruan et al. Trends Based on Biotechnol 22 (1): 23-30), the company was commissioned to Takara Bio Inc. (Kyoto).

1) Preparation of Tag library First, 20 ng of the small RNA fraction was treated with Bacterial Alkaline Phosphatase to remove the 5 'phosphate group, and then the DNA / RNA chimeric adapter (3' Adapter). Next, electrophoresis was performed on a 15% denaturing polyacrylamide gel, and the small RNA fraction to which the adapter was bound was separated and collected. At this time, a marker obtained by binding a 3 ′ adapter to 18-mer and 26-mer synthetic RNAs was used. Moreover, small RNA Gel Extraction Kit (TaKaRa Bio) was used for collection of the small RNA fraction from the acrylamide gel. The purified 3 ′ adapter-bound small RNA was treated with T4 DNA Polynucleotide Kinase to give a phosphate group to the 5 ′ end, and then a different DNA / RNA chimera adapter (5 ′ adapter) was bound to the 5 ′ side. Thereafter, reverse transcription was performed using a primer complementary to the 3 ′ adapter and reverse transcriptase, and cDNA was synthesized. Using this cDNA as a template, PCR was performed using primers complementary to the 5 ′ adapter and 3 ′ adapter. A dNTP mixture containing 5 methyl dCTP as a substrate was used. Next, using the Sfa NI site incorporated in both adapter sequences, a DNA fragment containing a small RNA-derived cDNA sequence (hereinafter referred to as the Signature sequence) was prepared by Sfa NI digestion, and a 20% native polyacrylamide gel was used. After electrophoresis, separation and collection were performed. The obtained DNA fragment was ligated in a fixed direction adjacent to the Tag sequence in the Tag vector. A part of the reaction solution was used to calculate the transformation efficiency (titer) of E. coli, and a plasmid DNA corresponding to titer = about 1.4 × 10 ⁶ was prepared as a Tag library. At the same time, 96 plasmid DNAs were sequenced for the obtained colonies, and the shape of the insert in the library was confirmed.

2) Preparation of Megaclone microbeads A portion containing Tag sequence and cDNA was amplified by PCR using plasmid DNA as a template. At this time, a 5 ′ fluorescently labeled primer and a 5 ′ Biotin labeled primer on the cDNA side were simultaneously used to obtain a mixed amplification product of the fluorescently labeled DNA fragment and the Biotin labeled DNA fragment. After digesting the Tag sequence side of each DNA fragment with the restriction enzyme Pac I, the Tag sequence part was made into a single strand by allowing T4 DNA polymerase to act in a reaction solution containing dGTP to prepare loadable DNA.

  Anti-Tag beads and loadable DNA were mixed, and each cDNA fragment was selectively bound to each bead by hybridizing the Tag sequence and the Anti-Tag sequence. After the beads loaded with DNA were adsorbed by Streptavidin magnetic beads, washing was performed while monitoring the fluorescence intensity to eliminate non-specific binding, and the beads bound with cDNA were purified.

  The purified and recovered beads fill the gap between the cDNA and the Anti-Tag using T4 DNA polymerase, form a covalent bond using T4 DNA ligase, and then digest with the restriction enzyme Dpn II. A vector-derived sequence containing a fluorescent group was removed, and a 2-stepper or 4-stepper adapter for MPSS was bound to each of the two to prepare Megaclone microbeads.

3) Reading of signature sequence by MPSS device
One flow cell was used for each of 2-stepper and 4-stepper, and approximately 1.3 × 10 ⁶ beads were filled in each flow cell. Set the flow cell in the analyzer for MPSS, repeat the enzyme reaction according to the method described in the reference (Brenner S. et al. Nature Biotech., 18: 630-634 (2000)), and the signature sequence on each Megaclone microbead. Reading up to 22 bases. That is, the steps of (1) Bbv I digestion into a single strand, (2) Encoded adapter binding, and (3) Decoder probe reading of the nucleotide sequence were repeated to bind the 4-stepper adapter. For flow cell beads, read a total of 22 bases in units of 4 bases from the 5 'end, and for flow cell beads to which a 2-stepper adapter is bound, shift the 4-stepper reading frame by 2 bases and read a total of 22 bases in units of 4 bases. It was. From the fluorescence image data of the beads hybridized with each Decoder probe, the fluorescence intensity of each bead is digitized, and a signature sequence that meets the standards for signal intensity, S / N ratio, presence / absence of bead deviation, etc. is judged to be valid. This was used for the subsequent analysis.

4) Extraction and filtering of signature sequence The portion corresponding to the adapter-derived sequence is removed from the 22-base sequence read from each flow cell, and low-reliability sequence data is filtered and removed. Converted to a number (tpm: transcripts per microllion). The results of MPSS are shown in Table 1, respectively. In the table, “Beads” indicates the number of beads (arrays) from which valid data was obtained in each flow cell, and “Signatures” indicates the type of signature array read.

(Example 2) Acquisition of microRNA from Eucalyptus In order to search for a candidate microRNA sequence from the signature sequence acquired in Example 1, secondary structure prediction of a neighboring sequence on the genome contig including the signature sequence was performed. . The signature sequence was mapped to the contig sequence and singlet sequence of the eucalyptus genome provided by Kazusa DNA Laboratory. Up to 2 base mismatches were allowed, and (1) upstream 128base and downstream 128base, (2) upstream 228base and downstream 228base genomic sequences were obtained from the mapped position. The contig sequence, singlet sequence and signature sequence used in the analysis are as follows.
Contig sequence: 17,311
-Singlet array: 21,880
・ Signature sequence: 29,783 (rRNA and other filtered sequences)
On the other hand, the 150 base and 250 base windows including the signature array were shifted every 5 bases, and secondary structure prediction was performed for the arrays included in each window. A sequence having the minimum free energy satisfying the following criteria was selected as a microRNA candidate. However, internal loop and bulge checks were performed on the loop side array from Signature.
-Has a stem-loop structure.
・ 22base including Signature sequence has 16 strands or more complementary strand.
・ Loop is 20 base or less.
-Internal loop is 10 base or less.
・ Bulge is 5 base or less.

A homology search was performed on known microRNAs to see if the signature sequence contained known microRNAs. The following database was used for information on known microRNAs.
miRBase v8.2 (http://microrna.sanger.ac.uk/)

  Table 2 shows the transition of the number of sequences in the analysis. There were a total of 751 sequences (SEQ ID NOs: 1 to 751) having structures satisfying the criteria.

(Example 3) Expression analysis of eucalyptus microRNA In order to investigate the expression level of the eucalyptus microRNA obtained in Example 2, analysis was carried out by Northern hybridization using eucalyptus meal as a material. Total RNA was extracted from eucalyptus meal in the same manner as in Example 1, and then small RNA was concentrated using mirVana miRNA Isolation Kit (Applied Biosystems). The small RNA was concentrated according to the method for isolating small RNA from the total RNA of the kit. Next, 10 μg of small RNA was electrophoresed on 15% TBE / Urea gel (Invitrogen), and blotted onto a hybridization membrane using a transblot SD cell semi-dry blotting apparatus (BioRad). As for the probe, four microRNA sequences (SEQ ID NOs: 297, 320, 546 and 263) were extracted from 751 total microRNAs obtained in Example 2 without having homology with known reports, and their complementary DNAs were artificially synthesized. , it was used as labeled with ^{Dig Oligonucleotide Tailing Kit, 2 nd Generation} ( Roche). For hybridization, Dig Easy Hyb (Roche) was incubated overnight in a 37 ° C solution of poly (A) (final concentration 0.1 mg / ml) and poly (dA) (final concentration 2 μg / ml). The 2 × SSC solution at 0 ° C. was washed twice as a washing solution. Thereafter, the probe was reacted with an alkaline phosphatase-labeled anti-dicoxygenin antibody and Fab fragment (Roche), and the luminescence obtained by the reaction with the chemiluminescent substrate CDP-Star (Roche) was analyzed by the LAS3000 system (Fuji Film). As a result, we succeeded in detecting the expression of all four microRNAs in Eucalyptus cocoon (FIG. 1). These microRNAs differ from those previously reported in plants, and it was revealed that novel microRNAs were expressed in Eucalyptus cocoons.

  By using the nucleic acid information of the present invention, in plants, particularly tree plants, for the purpose of promoting the growth, increasing the volume, or enhancing the stress tolerance, hypertrophy (growth), rooting It becomes possible to appropriately control flower buds and the like.

The hybridization result which shows having detected the expression of the microRNA of this invention is shown. Lanes 1 to 4 show the expression of microRNAs of SEQ ID NOs: 297, 320, 546 and 263, respectively.

Claims (19)

A nucleic acid selected from the group consisting of DNA and RNA of the following (a) to (h) or a nucleic acid combination consisting of a plurality of nucleic acids for use in controlling plant morphogenesis.
(a) DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 1 to 751
(b) DNA comprising a nucleotide sequence having at least 80% identity with any one of SEQ ID NOs: 1 to 751
(c) DNA complementary to the DNA of (a) above
(d) DNA complementary to the DNA of (b) above
(e) RNA encoding the DNA of (a) above
(f) RNA encoding the DNA of (b) above
(g) RNA encoding the DNA of (c) above
(h) RNA encoding the DNA of (d) above   A plant encoding a double-stranded DNA comprising any one of SEQ ID NOs: 1 to 751 or a base sequence having at least 80% identity with the base sequence and a base sequence complementary to the base sequence A nucleic acid, or a nucleic acid combination thereof, that is an siRNA for use in controlling the morphogenesis of the above.   The nucleic acid or nucleic acid combination according to claim 1 or 2, wherein the plant is a tree.   The nucleic acid or nucleic acid combination according to claim 3, wherein the tree is a Eucalyptus plant.   The nucleic acid or nucleic acid combination thereof according to claim 2, wherein the siRNA further comprises an overhang sequence at the 3 'end of the sense strand and the antisense strand.   The nucleic acid or nucleic acid combination thereof according to claim 2, wherein the siRNA further comprises a loop sequence between the sense strand and the antisense strand.   A vector comprising a DNA comprising any one of SEQ ID NOs: 1 to 751, or a base sequence having at least 80% identity with the sequence.   A transformed microorganism or transformed plant cell comprising the vector according to claim 7.   A transformed plant comprising the transformed plant cell according to claim 8.   The transformed plant according to claim 9, wherein the plant is a tree such as a Eucalyptus plant.   A nucleic acid according to any one of claims 1 to 6, or a nucleic acid combination thereof, or a vector according to claim 7, or a transformed plant cell or transformed microorganism according to claim 8. A method for controlling plant morphogenesis.   12. The method of claim 11, wherein the method is control of organ morphogenesis.   A microarray comprising a combination of nucleic acids selected from the group consisting of the nucleic acids of (a) to (h) according to claim 1.   The microarray according to claim 13, wherein the nucleic acid is RNA.   The microarray according to claim 13, wherein the nucleic acid is DNA.   The microarray according to any one of claims 13 to 15, which is used for screening for a substance that controls plant morphogenesis.   The microarray according to any one of claims 13 to 15, which is for identifying a gene involved in plant morphogenesis, a transcription product or a translation product thereof.   A seed derived from the transformed plant according to claim 9 or 10.   A progeny of the transformed plant according to claim 9 or 10.

Images