JP2004290064A - Gene inducing formation of lateral branch and protein encoded by the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To elucidate a molecular biological mechanism for the differentiation of a lateral bud into a stem to develop a technique for uniformly, stably and safely controlling the lateral branch formation of a plant, and to develop a technique by which an adventitious bud can generally, stably, uniformly and easily be induced from a cultured cell. <P>SOLUTION: A DNA containing a specific nucleotide sequence, a protein containing a specific amino acid sequence, a recombinant DNA structure containing the DNA, and a plant cell, tissue, organ, plant or its posterity transformed with the same are provided. A method for increasing or decreasing the lateral bud formation of a plant comprises increasing or decreasing the expression level of the DNA. A method for forming an adventitious bud from a cultured plant cell comprises transforming the cultured plant cell with a recombinant DNA structure. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００７７】
ＣＵＣ３遺伝子の特徴的な構造部分は、以下の通りである。

【００７８】
実施例３：突然変異部位の決定
上記で得られるＣＵＣ３遺伝子内の突然変異部位の決定は、本質的に製造者の指示にしたがって、パーキンエルマー社のＡＢＩ３１００ ＤＮＡシークエンサーを用いて、ｃｕｃ３変異体におけるＣＵＣ３遺伝子内の塩基配列が野生型のものと異なる部位を決定した。
ｃｕｃ３−１ 第一イントロンと第一エキソンの境目のＡＧ：ＧＴ−＞ＡＧ：ＡＴに変異、イントロンの切り出しができないと推定
ｃｕｃ３−２ １塩基置換により３０番目のプロリンがセリンに変化
ｃｕｃ３−３ １塩基置換により１０３番目のトリプトファンがストップコドンに変化ｃｕｃ３−４ １塩基置換により２５番目のグリシンがアスパラギン酸に変化
塩基配列の決定は、本質的に製造者の指示にしたがって、パーキンエルマー社のＡＢＩ３１００ ＤＮＡシークエンサーを用いて行う。
【００７９】
実施例４：酵母を使った転写活性化の検定
フォワードプライマー：５’−ＧＧＡＡＴＴＣＡＴＧＣＴＴＧＣＧＧＴＧＧＡＡＧＡＴＧＴ−３’（配列番号６）および
リバースプライマー５’−ＣＣＧＣＴＣＧＡＧＣＴＡＣＡＧＣＴＧＧＡＡＴＣＣＴＡＡＡＧＧ−３’（配列番号７）
をプライマーとして、Ｐｙｒｏｂｅｓｔ（宝酒造）を用いてＰＣＲでＣＵＣ３のＯＲＦを増幅する。鋳型には発芽７日目のシロイヌナズナの芽生え由来の全ＲＮＡを逆転写して得られるｃＤＮＡを用いる。ＰＣＲ産物をＥｃｏＲＩ／ＸｈｏＩで切断後、ｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）のＥｃｏＲＩ／ＸｈｏＩ部位にクローニングする。再びそのＥｃｏＲＩ／ＸｈｏＩ断片をｐＧＢＴ９のＥｃｏＲＩ／ＳａｌＩ部位にリクローニングし、ＣＬＯＮＴＥＣＨ社のｙｅａｓｔ ｐｒｏｔｏｃｏｌ ｈａｎｄｂｏｏｋに従って、酵母Ｙ１８７株に形質転換を行い、β−ガラクトシダーゼの活性を測定する。結果は、ＣＵＣ３が転写因子として機能することを指摘する。
【００８０】
【表２】

【００８１】
実施例５：形質転換カルスを使った不定芽形成能の検定
滅菌したシロイヌナズナの種子をＧＭ培地上に蒔き、発芽後寒冷紗の下で、弱光により胚軸を徒長させる。７日間培養の後、胚軸を切り出し、６日間ＣＩＭ培地上で培養しカルス化させる。このカルスをアグロバクテリウムをＯＤ６００＝ ０．２と成るよう懸濁したＢ５−ＭＥＳに浸しその後、ＣＩＭプレート上でアグロバクテリウムと２日間共培養する。次にカルスを洗浄液で１０回以上洗い、その後抗菌用の抗生物質の入ったＳＩＭ選択培地上で培養しシュート形成を誘導する。
上記のｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）にクローニングしたＣＵＣ３ ｃＤＮＡをＳｐｅＩ／ＸｈｏＩ断片として切り出し、ｐＤＨ１２３のＳｐｅＩ／ＸｈｏＩ部位にリクローニングし、アグロバクテリウムＧＶ３１０１：：ｐＭＰ９０に形質転換する。
【００８２】
ＧＭプレート
ムラシゲ−Ｓｋｏｏｇ塩 ４．３ ｇ／Ｌ
スクロース １０．０ ｇ／Ｌ
ミオイノシトール ０．１ ｇ／Ｌ
０．０５％ （ｗ／ｖ） ＭＥＳ−ＫＯＨ（ｐＨ５．７） ５．０ ｍｌ／ｌ
チアミン塩酸塩 ２０．０ μｇ／Ｌ
ニコチン酸 １．０ μｇ／Ｌ
ピリドキシン塩酸塩 １．０ μｇ／Ｌ
ビオチン １．０ μｇ／Ｌ
ゲランガム ５．０ ｇ／ｌ
【００８３】
カルス誘導培地（ＣＩＭ）
ガンボルグのＢ５塩 ３．３ｇ／Ｌ
グルコース ２０ｇ／Ｌ
ミオイノシトール ０．１ｇ／Ｌ
ＭＥＳ ＫＯＨ ０．５ｇ／Ｌ
チアミン塩酸塩 ２０．０ μｇ／Ｌ
ニコチン酸 １．０ μｇ／Ｌ
ピリドキシン塩酸塩 １．０ μｇ／Ｌ
ビオチン １．０ μｇ／Ｌ
２，４−Ｄ ０．５ｍｇ／Ｌ
カイネチン ０．１ｍｇ／Ｌ
ゲランガム ７．５ ｇ／Ｌ
【００８４】
シュート誘導培地（ＳＩＭ）
ガンボルグのＢ５塩塩 ３．３ｇ／Ｌ
グルコース ２０ｇ／Ｌ
ミオイノシトール ０．１ｇ／Ｌ
ＭＥＳ ＫＯＨ ０．５ｇ／Ｌ
チアミン塩酸塩 ２０．０ μｇ／Ｌ
ニコチン酸 １．０ μｇ／Ｌ
ピリドキシン塩酸塩 １．０ μｇ／Ｌ
ビオチン １．０ μｇ／Ｌ
ＩＡＡ ０．１５ ｍｇ／Ｌ
２ｉＰ ０．５ ｍｇ／Ｌ
ゲランガム ７．５ ｇ／Ｌ
【００８５】
Ｂ５−ＭＥＳ
ガンボルグのＢ５塩 ３．３ ｇ／Ｌ
ＭＥＳ ＫＯＨ ０．５ ｇ／Ｌ洗浄液
Ｂ５−ＭＥＳに２００ｍｇ／Ｌ バンコマイシン， ２００ｍｇ／Ｌ ｃｌａｆｏｒａｎ（セフォタキシム ナトリウム）を加えた。
選択培地はＳＩＭに１００ｍｇ／Ｌ ａｕｇｐｅｎｉｎ（カリウム ｃｌａｖｕｌａｎａｔｅ ： ｔｉｃａｒｃｉｌｌｉｎ ナトリウム ＝ １：１５）， ２０ｍｇ／Ｌ ハイグロマイシンＢを加えた。
結果はＣＵＣ３ ｃＤＮＡによる形質転換が、カルスから不定芽を誘導することを示している。
【表３】

【００８６】
【発明の効果】
本発明に記載の新規遺伝子を使用して、植物の側枝形成を制御する、分子生物学的手段が得られる。そしてこの手段により、植物育種において側枝形成特性が変化した植物系統を取得することが可能となると言う顕著な効果がもたらされる。また本発明の新規遺伝子を使用して、培養植物細胞から不定芽／不定根を安定的かつ容易に誘導することによって、同一遺伝子型のクローンを大量増殖することが可能となるという顕著な効果がもたらされる。
【００８７】
【配列表】

【図面の簡単な説明】
【図１】図１は、ｃｕｃ１ｃｕｃ２、ｃｕｃ１ｃｕｃ３およびｃｕｃ２ｃｕｃ３突然変異株のカップ形状子葉（ｃｕｐ ｓｈａｐｅｄ ｃｏｔｙｌｅｄｏｎ）を示す。
【図２−１】図２−１は、ＣＵＣ３のヌクレオチド配列を示す。
【図２−２】図２−２は、ＣＵＣ３のヌクレオチド配列を示す。
【図２−３】図２−３は、ＣＵＣ３のヌクレオチド配列を示す。
【図２−４】図２−４は、ＣＵＣ３のヌクレオチド配列を示す。
【図３】図３は、野生型およびｃｕｃ２ｃｕｃ３の２サブタイプの突然変異株シロイヌナズナを示す。野生型は正常に側芽を形成するのに対し、ｃｕｃ２−１ｃｕｃ３−２は側芽形成が抑制され、ｃｕｃ２−１ｃｕｃ３−１は側芽形成が完全に阻害された。
【図４】図４は、ＣＵＣ３タンパク質のアミノ酸配列を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to plant DNA, proteins and their uses.
[0002]
[Prior art]
In actual agricultural and forestry production sites, control of side buds, such as pruning and axillary bud removal, are difficult and labor intensive operations. Some attempts have been made to control this by chemical treatment, such as with plant growth regulators. However, it is not yet a technology that can achieve this uniformly, stably, and safely.
[0003]
Even within the same species, there are lines with high, few, or few side bud development. However, its genetic background remains unknown. And with regard to side bud formation characteristics, the breeding of lines with the desired trait remains difficult by means of clear breeding. Moreover, how the lateral buds differentiate from the stem is still unknown.
[0004]
Cell culture systems have been used to stably obtain large numbers of clones of the same genotype. In this system, the formation of adventitious buds from cultured cells is an essential process for regenerating plants from cultured cells, which is possible for some species by selecting plant regulators in the culture medium. However, there are still plants that cannot or do not induce adventitious buds from callus. It is still difficult to achieve this by general, uniform, stable and easy means.
[0005]
Thus, there is still a need for an approach that can control plant side buds in a general, stable and uniform manner. In the breeding plan, specifying genes related to the control of side buds enables plant breeding based on clear breeding goals. Furthermore, in order to efficiently produce large amounts of genetically uniform clones, it is desired to develop a system that can generally, stably, uniformly and easily induce adventitious buds from cultured cells.
[0006]
Conventionally, the present inventors have used CUC1 (CUP-SHAPED COTYLEDON 1) (see SEQ ID NO: 2) and CUC2 gene (SEQ ID NO: 3) that are involved in the formation of shoot apical meristem and the separation of organs during embryonic development using Arabidopsis thaliana. Or 4), all of which are polymorphic genes of the gene (see FIG. 1). In the case of the cuc1 or cuc2 single mutant, cotyledon fusion is observed at a very low frequency. On the other hand, the cuc1 cuc2 double mutant forms one cup-shaped cotyledon by raising the border of the cotyledon, and does not form a shoot apical meristem, and thus is a seedling lethal. CUC1 and 2 both encode proteins having a NAC domain and are thought to function as transcription factors.
[0007]
Aida et al. (1997) investigated a series of cuc mutants and isolated CUC1 and CUC2. These double mutants show complete inhibition of embryonic meristem meristem formation and partial inhibition of adventitious bud formation during shoot regeneration of calli from this double mutant. Has been described. In addition, it describes that the CUC1 and CUC2 genes appear to be involved in the formation of embryonic growth metastatic tissue and meristem in shoot regeneration. However, this double mutant can consistently explain that embryonic meristem meristems are completely deficient and that calli from this double mutant can form adventitious buds The mechanism is still unknown. Furthermore, there is no disclosure or suggestion of the involvement of the CUC1 and CUC2 genes in lateral bud formation. In a double mutant of a mutant (cuc1-6) and a CUC2 mutant (cuc2-1) having a weak functional deletion of the CUC1 gene, shoot apical meristem is formed. In this double mutant, side branches are formed normally (Hinohara, unpublished data). These results strongly suggest that the CUC3 gene, unlike the CUC1 and CUC2 genes, is involved in the lateral bud formation mechanism.
[0008]
Also, Aida et al. (1997) suggest that the CUC1 and CUC2 genes appear to be functionally overlapping genes, with mutants of either gene being phenotypically similar to each other and wild-type. It describes that it shows only a weak mutation in comparison (see Non-Patent Document 1). However, Aida (1997) does not disclose or suggest the CUC3 gene of the present invention. Furthermore, Aida et al. (1997) do not disclose or suggest any useful properties for a series of cuc mutants.
[0009]
Takada et al. Describe that the CUC1 gene and the CUC2 gene have high homology (see Non-Patent Document 2). However, Takeda et al. Do not describe the CUC3 gene of the present invention at all.
[0010]
Furthermore, Takada et al. Describe that overexpression of the CUC1 gene by the CaMV 35S promoter strongly promotes ectopic adventitious bud formation (see Non-Patent Document 2). In addition, it is described that the adventitious bud is mainly formed on cotyledons, and rarely on rosette leaves. However, Takada et al. Does not disclose or suggest any side bud formation properties of a series of mutant strains.
[0011]
Daimon et al. Disclose that overexpression of the CUC1 or CUC2 gene promotes adventitious bud formation in calli derived from hypocotyls of wild-type Arabidopsis thaliana (see Non-Patent Document 3).
[0012]
Also, Daimon et al. Describe that transformation with CUC1 and CUC2 genes promotes shoot formation from callus (see Non-Patent Document 3). However, Daimon et al. Do not disclose or suggest the CUC3 gene.
[0013]
[Non-patent document 1]
Mitsuhiro Aida et al. , "Genes Involved in Organ Separation in Arabidopsis: An Analysis of the Cup-Shaped Coiledon Mutant", The Plant Cell, Vol. 9, pp. 841-857 (1997).
[Non-patent document 2]
Shinobu Takada et al. , "The CUP-SHAPEED COTYLEDON1 gene of Arabidopsis regu- lates short applica- tional product formation", Development, Vol. 128, pp. 1127-1135 (2001).
[Non-Patent Document 3]
Yasufumi Daimon et al. , "The CUP-SHAPED COTYLEDON Genes Promote Adventious Shot Formation on Calli", Plant Cell Physiol. Vol. 44, pp. 113-121 (2003).
[Non-patent document 4]
Mitsuhiro Aida et al. , "Shoot apical meristem and cotyledon formation dur- ing Arabidopsis embryogenesis: interaction ammonia the CUP-SHAPED COTYLEDONSME STOREV and SHOEMOVESMESHOTESV. 126, pp. 1563-1570 (1999).
[0014]
[Problems to be solved by the invention]
It is an object of the present invention to provide genes that regulate lateral bud formation in plants, proteins encoded thereby, and their uses.
[0015]
[Means for Solving the Problems]
The inventors treated the cuc2 mutant with EMS to isolate new genes involved in the formation of cotyledon borders and shoot apical meristems. Has been isolated. However, when the obtained cuc2 enhancer mutant was examined genetically, it was found to be mostly new alleles of CUC1.
[0016]
As a result of intensive studies on the remaining mutants, the inventors have found a mutation in the NAC box gene belonging to the same family as CUC1 and CUC2. The present inventors have now succeeded in isolating this novel gene. This new gene is named CUC3 (SEQ ID NO: 1, see FIG. 2). A double mutant of CUC1 and CUC2 shows a cuc phenotype, whereas a mutant of only CUC1 and CUC2 does not show a cuc mutant phenotype (see Non-Patent Document 1, supra). However, the mutant strain in which the CUC3 gene and the CUC1 gene are mutated shows a cuc mutation. The mutant strain in which the CUC3 gene and the CUC2 gene are mutated also shows a cuc mutation.
[0017]
CUC1 and CUC3 double mutants or CUC2 and CUC3 double mutants, in addition to inhibiting cotyledon (embryonic) meristems, also show that these mutants It was found that side branch formation, especially side branch growth point formation, was substantially inhibited (see FIG. 3). These findings are surprising in view of the fact that no genes affecting the formation of side branch meristems have been isolated.
[0018]
Thus, the present invention:
(A) the nucleotide sequence of SEQ ID NO: 1,
(B) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added,
(C) the complementary sequence of (a) or (b), and
(D) a nucleotide sequence capable of hybridizing to any of the nucleotide sequences (a) to (c) under stringent hybridization conditions;
A DNA comprising a nucleotide sequence selected from the group consisting of:
[0019]
(A) SEQ ID NO: 1 positions 1 to 205, 338 to 612, 1283 to 1807, 64 to 205, 338 to 612, 338 to 612, 1283 to 1315, 1 to 23, 1786 to A nucleotide sequence represented by position 1807 or 1345-1364;
(B) a nucleotide sequence encoding the amino acid sequence of positions 22 to 171 of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added,
(C) the complementary sequence of (a) or (b), and
(D) a nucleotide sequence capable of hybridizing to any of the nucleotide sequences (a) to (c) under stringent hybridization conditions;
A DNA comprising a nucleotide sequence selected from the group consisting of:
[0020]
A protein comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added;
A protein comprising the amino acid sequence of positions 22 to 171 of SEQ ID NO: 5, or the amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added;
A recombinant DNA construct comprising the DNA;
A plant cell, tissue, organ, plant or progeny transformed with said recombinant DNA construct;
A method for substantially increasing or decreasing lateral bud formation of a plant as compared to a control plant, wherein the expression level of the DNA is substantially increased or decreased as compared to a control plant. how to;
[0021]
The method further comprising transforming a plant with a recombinant DNA construct comprising the DNA, a promoter operably linked thereto, and a suitable transcription terminator as desired;
The method further comprising transforming a plant with a recombinant DNA construct comprising a sequence that reduces the expression of said DNA, a promoter operably linked thereto, and a suitable transcription terminator as desired;
A method of forming adventitious buds from the cultured plant cells by transforming the cultured plant cells with the above-mentioned recombinant DNA construct;
[0022]
A method for producing a plant, comprising transforming a cultured plant cell with the recombinant DNA construct to form an adventitious bud from the cultured plant cell;
A plant cell, tissue, organ, plant or progeny obtained by the above-mentioned production method;
I will provide a.
[0023]
The protein preferably contains the amino acid sequence at positions 22 to 171 of SEQ ID NO: 5, which is a NAC domain region.
Preferred variants of the above proteins include, for example, those in which glutamic acid at position 69 of SEQ ID NO: 5 has been changed to aspartic acid, asparagine, isoleucine, leucine, tyrosine, or serine.
[0024]
Preferred variants of the above proteins include, for example, those in which proline at position 30 of SEQ ID NO: 5 has been mutated to serine.
Preferred mutants of the above protein include, for example, those containing tryptophan at position 103 of SEQ ID NO: 5 and deletions after tryptophan.
Preferred variants of the above proteins include, for example, those in which glycine at position 25 of SEQ ID NO: 5 has been mutated to aspartic acid.
DNA comprising a nucleotide sequence encoding the preferred mutant protein is also included in the present invention.
[0025]
The DNA (CUC3 gene) of the present invention has a function of substantially changing, for example, increasing or decreasing the formation of side branches of a plant as compared with a control plant by increasing or decreasing its expression level. .
The DNA of the present invention preferably corresponds to the case where the expression level of the DNA represented by the nucleotide sequence of SEQ ID NO: 2 (CUC1 gene) or the DNA represented by the nucleotide sequence of SEQ ID NO: 3 (CUC2 gene) increases or decreases. By increasing or decreasing the expression level of the DNA of the present invention, side branch formation of the plant has a function of substantially changing, for example, increasing or decreasing, as compared with the control plant.
[0026]
More preferably, the DNA of the present invention is obtained by increasing or decreasing the expression level of the DNA represented by the nucleotide sequence of SEQ ID NO: 2, thereby increasing or decreasing the expression level of the DNA of the present invention. Has a function of substantially changing, for example, increasing or decreasing, as compared to a control plant.
The DNA of the present invention is most preferably obtained by increasing or decreasing the expression level of the DNA represented by the nucleotide sequence of SEQ ID NO: 3, thereby increasing or decreasing the expression level of the DNA of the present invention. Has a function of substantially changing, for example, increasing or decreasing, as compared to a control plant.
Altering, eg, increasing or decreasing the expression level of the DNA of the present invention alters, eg, increases, lateral bud formation of not only Arabidopsis but also other plants, such as dicotyledonous plants, monocotyledonous plants, gymnosperms, angiosperms. Or it can be said that it can be reduced.
[0027]
As described in Example 4, when the DNA of the present invention was introduced into eukaryotic cell yeast, GUS activity was increased. This indicates that the protein encoded by the DNA of the present invention can function as a transcription factor.
[0028]
The term "one to ten or more amino acids are substituted, added or deleted" when used in reference to the amino acid sequence of a protein is 1, 2, 3, 4, 5, 6, 7, 8, 9, Alternatively, it means that 10 amino acids have been substituted, added or deleted. The protein having an amino acid sequence in which one or several amino acids are substituted, added or deleted "preferably has a function substantially similar to the protein having the amino acid sequence of SEQ ID NO: 5.
[0029]
"Stringent hybridization conditions" and "stringent hybridization wash conditions" are nucleic acid hybridization experiments, such as Southern and Northern hybridizations, that are sequence dependent and will be different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures.
[0030]
Detailed guide to the hybridization of nucleic acids, Tijissen (1993) Laboratory Techniques in Biotechemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2''Overview of principles of hybridization and the strategy of nucleic acid probe assays '' Elsevier , New York. In general, highly stringent hybridization and washing conditions are determined by the thermal melting point (T.sub.T) for a particular sequence at a defined ionic strength and pH. _m ) Is selected to be less than about 5 ° C. Typically, under "stringent conditions", a probe will hybridize to its target sequence but not to other sequences.
[0031]
T _m Is the temperature (at a defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The very stringent conditions are the T _m Is chosen to be equivalent to Examples of stringent hybridization conditions for hybridization of complementary nucleic acids, having more than 100 complementary residues on a filter in a Southern or Northern blot, are hybridizations performed overnight, 50% formamide, 42 ° C. with 1 mg of heparin. An example of highly stringent hybridization wash conditions is 0.15 M NaCl, 72 ° C. for about 15 minutes. An example of stringent wash conditions is 0.2 × SSC wash, 65 ° C., 15 minutes (see Sambrook, supra for description of SSC buffer).
[0032]
Often, a low stringency wash is performed before a high stringency wash to remove background probe signals. An example of a moderate stringency wash, eg, for a double helix of more than 100 nucleotides, is 4-6 × SSC, 40 ° C., 15 minutes. For short probes (eg, about 10 to 50 nucleotides), stringent conditions typically include salt concentrations below about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other Salt), pH 7.0 to 8.3, and the temperature is typically at least about 30 ° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in a particular hybridization assay indicates the detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
[0033]
The following is an example of a set of hybridization / wash conditions that can be used to clone a homologous nucleotide sequence that is substantially identical to a reference nucleotide sequence of the present invention: Indicates that the reference nucleotide sequence contains 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ In 1 mM EDTA, 50 ° C., wash with 2 × SSC, 0.1% SDS, 50 ° C., more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ In 1 mM EDTA, 50 ° C., wash with 1 × SSC, 0.1% SDS, 50 ° C., more preferably still 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ In 1 mM EDTA, 50 ° C., wash at 0.5 × SSC, 0.1% SDS, 50 ° C., preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ In 1 mM EDTA, 50 ° C., wash with 0.1 × SSC, 0.1% SDS, 50 ° C., more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ Washing is performed in 1 mM EDTA at 50 ° C. at 0.1 × SSC, 0.1% SDS at 65 ° C.
[0034]
A recombinant DNA construct is a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, optionally functionally linked to a desired nucleotide sequence operably linked to a termination signal. Means a sequence containing a promoter that is operably linked. It typically also contains the sequences required for the correct translation of the nucleotide sequence. The recombinant DNA construct containing the desired nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. Recombinant DNA constructs can also be obtained in naturally occurring but useful recombinant form for heterologous expression. Typically, however, the recombinant DNA construct is heterologous to the host, i.e., the particular nucleic acid sequence of the recombinant DNA construct is not naturally present in the host, and upon transformation events, the host cell or ancestors of the host cell. Must have been introduced in
[0035]
Expression of the nucleotide sequence of the recombinant DNA construct may be under the control of a constitutive promoter, or of an inducible promoter that initiates transcription only when the host cell is exposed to certain external stimuli. In the case of a multicellular organism, such as a plant, the promoter can also be specific for a particular tissue, or organ, or stage of development.
[0036]
The DNA of the present invention is inserted into a suitable vector by inserting the DNA of the present invention into a plant cell, and then the transformed plant cells are regenerated into plant tissues, organs, and plants. The transformed plant cells are preferably those that can be regenerated into plant tissues, organs, and plants.
[0037]
The process of incorporating the DNA of the present invention into plant cells can be performed using conventional recombinant DNA technology. Such a technique is found, for example, in Shujunsha, Plant Cell Engineering Series 4, “Model Plant Experimental Protocol”. Generally, this involves inserting the DNA sequence of the invention into an expression system in which the DNA is heterologous (ie, not normally present) using standard cloning techniques known in the art. This recombinant DNA construct contains the necessary elements for the transcription and translation of the inserted protein coding sequence. Many art-known vector systems can be used, for example, plasmids, bacteriophage viruses, and other modified viruses. Suitable vectors are viral vectors, for example, the lambda vector system, λgt11, λgt10, and Sharon4; plasmid vectors, for example, pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK233-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems. The transformed cells are regenerated into whole plants, such that a change in the level of expression of the DNA of the present invention results in a change, eg, an increase or decrease, in the lateral bud formation properties of the transgenic plant.
[0038]
Plants to be transformed according to the present invention are monocotyledonous or dicotyledonous plants, maize, wheat, barley, rye, sweet potato, kidney bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish. , Spinach, asparagus, onion, garlic, capsicum, celery, pumpkin, hemp, zucchini, apple, pear, quince, melon, watermelon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple , Avocado, papaya, mango, banana, soybean, tomato, sorghum, sugar cane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis Na and trees plants, for example, softwood, for example, cedar, cypress, hardwood, but may include evergreen and deciduous without limitation. Once the desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species or transferred to other varieties / breeds of the same species, especially commercial varieties / breeds, using traditional breeding techniques. .
[0039]
In a preferred embodiment, for expression of a DNA of the present invention in a transgenic plant, a recombinant DNA construct can be constructed in which the DNA of the present invention is linked to a suitable promoter so as to function in the plant. The choice of promoter will vary depending on the time and location requirements for expression, and also on the target species. Thus, expression of the nucleotide sequences of the present invention in leaves, ears, inflorescences (eg, spikes, panicles, cobs, etc.), roots, and / or seedlings is preferred. Although many promoters from dicots have been shown to be functional in monocots and vice versa, ideally, dicot promoters should be used for expression in dicots, and A cotyledon promoter is selected for expression in monocotyledonous plants. However, there is no limitation on the origin of the selected promoter; it is sufficient that it be functional to drive expression of the nucleotide sequence in the desired cell.
[0040]
Preferred promoters for constitutive expression may include promoters from actin or ubiquitin encoding DNA and the CaMV 35S and 19S promoters. Other promoter sequences can include, but are not limited to, for example, chemically regulated promoters, wound-inducible promoters, green tissue-specific promoters, root-specific promoters, stem-specific promoters, and flower-specific promoters.
[0041]
In addition to selecting a suitable promoter, the constructs for expression of the DNA of the present invention in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence, if desired. Several such terminators are available and known in the art (eg, tml from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of the present invention.
[0042]
Many other sequences can be incorporated into the recombinant DNA constructs described in the present invention. These include sequences known to enhance expression, such as intron sequences (eg, from Adhl and bronze1) and viral leader sequences (eg, from TMV, MCMV and AMV).
[0043]
Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, a binary vector or vector having at least one T-DNA border sequence is suitable, while for direct gene transfer methods any vector is suitable, and Linear DNA containing only the desired construct may be preferred. For direct gene transfer methods, transformation with a single DNA species or co-transformation can be used (Schocher et al., Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transformation, transformation is usually (but not necessarily) performed with an antibiotic (kanamycin, hygromycin, or methotrexate), or a herbicide (Vasta). A selectable marker that can provide resistance to is contemplated.
[0044]
Examples of such markers include neomycin aphosphotransferase, hygromycin phorphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2,2-dichloropropionate dehalogenase, acetohydroxyacid synthase, 5-enolpyruvir-shikimate. -Phosphate synthase, haloaryl nitrilase, protoporphyrinogen oxidase, acetyl coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyltransferase, and β-glucuronidase. However, selection for plant transformation or selection of screening markers is not critical to the invention.
[0045]
Transformation of dicotyledonous plants
Techniques for transforming dicots are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium techniques involve direct uptake of exogenous genetic material by protoplasts or cells. This can be achieved by PEG or electroporation mediated uptake, particle bombardment mediated delivery, or microinjection. Examples of these techniques are described in Paszkowski et al. , EMBO J3: 2717-2722 (1984); Potrykus et al. , Mol. Gen. Genet: 199: 169-177 (1985); Reich et al. , Biotology 4: 1001-1004 (1986); and Klein et al. , Nature 327: 70-73 (1987). In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.
[0046]
Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledons due to its high transformation efficiency and widespread use for many different species. Agrobacterium transformation typically involves transferring a binary vector (eg, pCIB200 or pCIB2001) with the foreign DNA of interest into a host Agrobacterium strain (eg, pCIB200) either on the co-located Ti plasmid or on the chromosome. And transfer to a suitable Agrobacterium strain which may depend on complementation of the vir gene carried by strain CIB542 (Uknes et al., Plant Cell 5: 159-169 (1993)) for pCIB2001. The transfer of the recombinant binary vector into Agrobacterium is carried out in E. coli having the recombinant binary vector, having a plasmid such as pRK2013, and using the recombinant binary vector as a target Agrobacterium strain. Achieved by a three-way crossover method using a transferable helper E. coli strain, or alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hoefgen and Willmitzer). , Nucl. Acids Res. 16: 9877 (1988)).
[0047]
Transformation of a target plant species with a recombinant Agrobacterium usually involves co-culturing Agrobacterium with explants derived from the plant and follows protocols well known in the art. The transformed tissue is regenerated on a selective medium having an antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.
[0048]
Another approach to transforming plant cells with certain DNA involves forcing inert or biologically active particles into plant tissues and cells. All of this technology is disclosed in Sanford U.S. Patent Nos. 4,945,050, 5,360,006, and 5,100,792. Generally, the method involves driving the inert or bioactive particles into the cell under conditions effective to penetrate the outer surface of the cell and effect internalization. When using inert particles, the vector can be introduced into cells by coating the particles with a vector containing the desired DNA. Alternatively, the target cell may be surrounded by the vector so that the trace of the particle carries the vector into the cell. Bioactive particles (eg, dried yeast cells, dried bacteria or bacteriophage, each containing the DNA to be introduced) can also be driven into the plant cell tissue.
[0049]
Transform monocotyledonous plants
Transformation of most monocotyledonous species is also now routine. Preferred techniques include direct introduction into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformation can be contemplated with a single DNA species or multiple DNA species (ie, co-transformation), both of which are suitable for use in the present invention. Co-transformation may have the advantage of avoiding complete vector construction and creating transgenic plants having loci that are not linked for the desired and selectable marker genes, thereby providing If so, it becomes possible to remove the selectable marker in subsequent generations. However, a disadvantage of using co-transformation is that the frequency of separate DNA species being integrated into the genome is less than 100% (Schocher et al. Biotechnology 4: 1093-1096 (1986)). ).
[0050]
Patent applications EP 0292435, EP 0392225, and WO 93/07278 disclose the preparation of callus and protoplasts from selected inbred maize, transformation of protoplasts using PEG or electroporation, and transformation of maize plants from the transformed protoplasts. Describes techniques for regeneration. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) discloses a technique for the transformation of a corn line derived from A188 using particle bombardment. Further, WO93 / 07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describes a technique for transforming selected inbred maize by particle bombardment. This technique utilizes a 1.5-2.5 mm long immature corn embryo cut from corn ears 14-15 days after pollination and a PDS-1000 He Biolytics device for bombardment.
[0051]
Transformation of rice can also be performed by direct gene transfer techniques utilizing protoplasts or particle bombardment. Transformations mediated by protoplasts have been described for the Japonica and Indica types (Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Data, et al., Biotechnology 8, 736-740 (1990)). Both types can be transformed by a conventional method using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)). Further, WO 93/21335 describes a technique for transforming rice by electroporation.
[0052]
Patent application EP0332581 describes techniques for the production, transformation and regeneration of Poideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, the transformation of wheat using particle bombardment into cells of type C long-term renewable callus has been described by Vasil et al. (Biotechnology 11: 1553-1558 (1993)), and the transformation of wheat using particle bombardment of immature embryos and calli derived from immature embryos was performed by Vasil et al. (Biotechnology 10: 667-674 (1992)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)). However, a preferred technique for wheat transformation involves the transformation of wheat by particle bombardment of immature embryos and involves either a high sucrose or high maltose stage prior to gene delivery. Prior to bombardment, for induction of somatic embryos, MS medium containing 3% sucrose and 3 mg / L of 2,4-D (Murashiga and Skoog, Physiological Plantarum 15: 473-497 (1962)) is optional. Number of embryos (0.75-1 mm long) are plated and allowed to proceed in the dark. On the day of the selected bombardment, the embryos are removed from the induction medium and placed on a osmotic modulator (ie, an induction medium containing the desired concentration, typically 15% sucrose or maltose).
[0053]
The embryos are protoplasmically separated for 2-3 hours and then bombarded. Twenty embryos per target plate are typical, but not critical. Plasmids containing the appropriate gene (eg, pCIB3064 or pSG35) are precipitated on micrometer-sized gold particles using standard methods. Each plate of embryos is shot with a DuPont Biolytics ™ helium device using a burst pressure of 〜1000 psi, a standard 80 mesh screen. After bombardment, the embryos are returned to the dark and allowed to recover for approximately 24 hours (still on the osmotic modulator). After 24 hours, the embryos are removed from the osmotic modulator and returned to the induction medium, where they are held for about one month before regeneration. Approximately one month later, the explants of the embryos that develop the developing embryogenic calli further contain the appropriate selection agents (10 mg / L basta for pCIB3064 and 2 mg / L methotrexate for pSOG35). Transfer to regeneration medium (MS + 1 mg / L NAA, 5 mg / L GA). After approximately one month, the developed shoots are transferred to a larger sterile container known as "GA7" containing half strength MS, 2% sucrose, and the same concentration of selective agent.
Gene transfer of monocotyledonous plants using Agrobacterium is also common practice today. For a disclosure of the transformation of monocotyledonous plants using Agrobacterium see WO 94/00977 and US Pat. No. 5,591,616, which are incorporated herein by reference.
[0054]
Plastid transformation
Nicotiana tabacum c. v. 'Xanthinb' seeds were germinated seven per plate in a 1 inch circular array on T agar and essentially as described (Svab, Z. and Maliga, P. (1993) PANS 90, 913-917). ), 1 μm tungsten particles (M10, Biorad, Hecules, Calif.) Coated with DNA from plasmids pPH143 and pPH145, bombarded 12-14 days after seeding. Bombarded seedlings were incubated on T medium for 2 days, after which the leaves were excised and lighted (350-500 μmol photons / m ² / S) with the abscissa side up, RMOP medium containing 500 μg / ml spectinomycin dihydrochloride (Sigma, Wt. Louis, MO) (Svab, Z., Hajdukiewiczz, P. and Maliga, P .; 1990) Place on a plate of PNAS 87, 8526-8530. Resistant shoots that appear under blanched leaves after 3 to 8 weeks of bombardment are subcloned on the same selection medium, allowing callus to form, and Secondary shoots are isolated and subcloned: Complete isolation (homoplasmicity) of the transformed plastid genomic copy in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al., (1989)). Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor).
[0055]
BamHI / EcoRI digested total cellular DNA (Mettler, IJ (1987) Plant Mol Biol Reporter 5, 346-349) was separated on a 1% trisborate (TBE) agarose gel and nylon membrane (Amersham). And corresponding to a 0.7 kb BamHI / HindIII DNA fragment from pC8 containing part of the rps7 / 12 plastid targeting sequence. ³² Search with P-labeled random primed nucleotide sequence. Homoplasmic shoots are aseptically rooted on MS / IBA medium containing spectinomycin (McBride, KE et al., (1994) PNAS 91, 7301-7305) and transferred to a greenhouse.
[0056]
The plants obtained by transformation with the nucleic acid sequences of the present invention can be any of a wide variety of plant species, including monocotyledonous and dicotyledonous plants, but the plants used in the methods of the present invention are preferably It is selected from the list of target crops of agricultural importance mentioned above. Expression of the DNA of the present invention, combined with other properties important for production and quality, is incorporated into progeny plants through breeding. Breeding approaches and techniques are known in the art. For example, Welsh J. et al. R. See Fudamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1981); Singh, D., The Theory of Plant Breeding, 2nd Edition, Clarendon Press, Oxford (1987); P. , Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); Wricke and Weber, Quantitative Departures from the Governance and the Future. , Berlin (1986).
[0057]
A plant tissue, organ, plant, or progeny transformed with a recombinant DNA construct of the present invention has a substantially altered, such as increased or decreased, side bud formation property, particularly side bud meristem formation, as compared to a control plant. Are preferred.
[0058]
Increasing or decreasing the expression level of the DNA (CUC3 gene) of the present invention may preferably correspondingly increase or decrease the expression level of the CUC1 gene and / or CUC2 gene. Increasing or decreasing the level of expression of a particular DNA in a plant can be performed using means known in the art. Means for increasing the expression level of a particular DNA can include, but are not limited to, the use of a constitutive promoter, increasing the copy number of the DNA.
[0059]
Methods for increasing the expression of a particular DNA in a plant include transforming a plant cell with a separate recombinant DNA construct, eg, fusing the gene in a sense orientation with a constitutive promoter capable of strongly driving transcription of the gene. Generating a chimeric gene, which is expressed in a target plant cell in an expression cassette. The separate recombinant DNA construct may optionally contain multiple copies of the gene. Constitutive promoters suitable for expression in plants can include, but are not limited to, promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters.
[0060]
Means for reducing the expression level of a particular gene in a plant include mutagenesis of the endogenous CUC3 gene itself, eg, treatment with a mutagen, site-directed mutagenesis, mismatch repair, in vitro mutagenesis. Mutating the endogenous CUC3 gene itself and transforming plant cells with a separate recombinant DNA construct, such as transforming a plant cell with a recombinant DNA construct that contains sequences that reduce the level of expression of a particular gene, It may include, but is not limited to.
[0061]
Methods for reducing the expression of a particular gene in a plant include sequences that reduce the level of expression of the gene, e.g., antisense technology using antisense sequences, cosuppression technology using sense sequences, and ribozyme-encoding sequences. It may include the ribozyme approach used. The antisense effect was first demonstrated by Ecker et al. Using a transient gene expression method, in which antisense RNA introduced by electroporation exhibited an antisense effect in plants (JR Ecker and R). W. Davis, (1986) Proc. Natl. Acad. USA. 83: 5372). Thereafter, it has been reported that the expression of the target gene is reduced by the expression of antisense RNA in tobacco, petunia, and tomato, and is now established as a means of suppressing gene expression in plants.
[0062]
Cosuppression techniques take advantage of the phenomena caused by transformation of DNA having a sequence identical or similar to the target gene sequence. Cosuppression refers to a phenomenon in which when a gene having the same or similar sequence as a target gene is introduced into a plant by transformation, the expression of both the foreign gene and the target gene to be introduced is suppressed. Although the details of the mechanism of cosuppression are not clear, it is an effect often observed in plants (Curr. Biol. 7: R793, 1997; Cuur. Biol. 6: 810, 1996).
[0063]
Ribozymes are RNA molecules having catalytic activity. There are ribozymes having various activities. Among them, studies on ribozymes as RNA-cleaving enzymes have made it possible to design ribozymes for site-specific cleavage of RNA. Ribozymes include those having a size of 400 nucleotides or more, such as the group I intron type and M1 RNA contained in RNaseP, and those having an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, (1990) Protein nucleic acid enzyme, 35: 2191). In the present invention, the transcript of the target gene can be specifically cleaved using such a ribozyme to suppress the expression of the gene.
[0064]
Methods for mutagenizing plant cells can be performed according to any technique known in the art. The method may use a chemical, for example, a chemical mutagen selected from the group consisting of ethyl methanesulfonate (EMS), methyl-N-nitrosourea (MNU), and bleomycins. The method can also be practiced by irradiating radiation selected from the group consisting of UV, γ-ray, X-ray, and fast neutron.
[0065]
The definitions of terms used in this specification are as follows.
[0066]
Gene: The term "gene" is used broadly to mean any segment of DNA associated with a biological function. Thus, genes include coding sequences and / or regulatory sequences required for their expression. Genes also include, for example, unpredictable segments of DNA that form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a desired source or synthesis from known or predicted sequence information.
"Expression" of a gene means the transcription and / or translation of an endogenous gene or an exogenous, heterologous gene and / or a transgene. In the case of an antisense construct, for example, expression may mean only transcription of the antisense DNA.
[0067]
A "plant" is any plant at any stage of development, especially a seed plant, for example, a monocot or dicot. "Plant" also refers to a part of a plant, such as a leaf, stem, root, flower or flower part, fruit, pollen, egg cell, zygote, seed, cutting, cell, or tissue culture or any other plant. It can include parts or products.
The "progeny" refers to any plant produced by sexual or asexual reproduction from a plant, such as seeds, seedlings, shoots, bulbs, tubers, rhizomes, or a traditional propagation and / or breeding method from a plant. And any plant obtained by the application of
"Plant cells" are the structural and physiological units of plants, including protoplasts and cell walls. Plant cells can be in the form of isolated single cells or cultured cells, or as a highly organized unit, such as a plant tissue, plant organ, or part of a whole plant.
[0068]
"Cultured plant cells" refers to cultures of plant units, for example, protoplasts, cell culture cells, cells of plant tissues, pollen, pollen tubes, ovules, embryo sac, zygotes, and embryos at various stages of development Means culture.
[0069]
A "plant organ" is a distinct and visually composed and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.
As used herein, the term "adventitious bud" means all but the shoots of a plant. For example, shoots formed on leaves, stems or callus of plants are adventitious shoots. Here, the term “sprouts” refers to shoots that occur at certain sites, such as apical buds, axillary buds, and accessory buds.
As used herein, the term "adventitious root" means a root that is formed secondary from organs other than the root, such as callus.
As used herein, the term "side bud" may include buds that develop lateral to the stem axis, such as, but not limited to, axillary buds, primary buds, and accessory buds.
“To form side buds” means that the side buds, which could not be visually or microscopically visible at first, become visible.
"Substantially increase or decrease side bud formation as compared to control plants" refers to conventional statistical techniques, such as t-test, analysis of variance, multivariate analysis, as compared to side bud formation of control plants. For example, the lateral bud formation, especially the lateral bud growth point formation, was significantly detected at a significance level of 10%, 5%, 3%, 1%, 0.05%, and 0.01% by Duncan's multiple test. Means increasing or decreasing.
[0070]
A "promoter" is an untranslated nucleotide sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of DNA. The promoter region can also include other elements that act as regulators of gene expression.
"Protoplasts" are isolated plant cells that lack the cell wall or have only a portion of the cell wall.
[0071]
"Gene mutation" includes any detectable change in the nucleotide sequence of DNA. Examples of gene mutations can include, but are not limited to, point mutations, deletions, duplications, inversions, insertions, translocations, and the like.
[0072]
“Transformation” is a process for introducing a heterologous nucleic acid into a host cell or organism. In particular, "transformation" refers to the stable incorporation of a DNA molecule into the genome of a desired organism. A transformed cell, tissue or plant is understood to include not only the end product of the transformation process, but also its transgenic progeny.
[0073]
In addition, the meanings of the terms in this specification should be interpreted as meanings that can be generally used by those skilled in the art to which the present invention pertains, except for the prominent terms. In addition, gene introduction, mutation treatment, determination of a base sequence, and germination, growth, and management of a plant can be performed by any method known to those skilled in the art to which the present invention belongs (Sambrook et al., Molecular Cloning). : A Laboratory Manual, 2nd ed. Cold Spring Harbor (1989)).
[0074]
【Example】
Example 1 Induction of Mutants
A cuc2-1 mutant of the Arabidopsis thaliana ecotype Ler strain is obtained essentially as described by Aida et al., 1997, supra. The resulting cuc2-1 mutant was purified from Fukiki, H .; et al. , Plant Physiol. 110, 933-943 (1991), under constant white light and under controlled conditions of 23 ° C. A new mutation (cuc3-1, -2, -3, -4) is induced by treating the seeds of this cuc2-1 mutant with ethyl methanesulfonic acid (EMS). The EMS process is performed in the draft according to the following procedure. To a 15 ml Falcon tube, 8 μl of EMS solution is added to 2 ml of B5 (3.3 g / l mixed salt for Gamborg B5 medium) and the composition is shown below, and the mixture is stirred well. About 5000 seeds of the cuc2 mutant are added and left at room temperature for 8 hours. Thereafter, the seeds are washed 10 times with the B5 solution.
[0075]
Example 2: Isolation of CUC3 gene
The CUC3 gene is isolated by the positional cloning method. Specifically, it is carried out in accordance with Shujunsha Plant Cell Engineering Series 14 “Plant Genome Research Protocol” 2-5-b “Map-Based Cloning of Arabidopsis” (p95-102).
[0076]
"mapping"
The wild-type Cuc3-1 and cuc2-1 of Ler are crossed with the wild-type Col to select those showing the cucu2cuc3 phenotype in the F2 generation, and the DNA is independently extracted from each individual by an ordinary method. In the first mapping, when about 40 chromosomes were examined using DNA markers showing polymorphism in Col and Ler, it was found that NGA692 on chromosome 1 was strongly linked and CUC3 was located on chromosome 1. . Subsequently, further examination of about 40 chromosomes reveals that At1g76420 has a mutation.
The nucleotide sequence is determined using a Perkin Elmer ABI3100 DNA sequencer essentially according to the manufacturer's instructions. The results are as follows.
Structure of CUC3 gene
[Table 1]

[0077]
The characteristic structural parts of the CUC3 gene are as follows.

[0078]
Example 3: Determination of mutation site
The determination of the mutation site in the CUC3 gene obtained above was carried out essentially using the Perkin Elmer ABI3100 DNA sequencer according to the manufacturer's instructions, in which the nucleotide sequence in the CUC3 gene in the cuc3 mutant was wild-type. A different site was determined.
cuc3-1 Mutation of AG: GT-> AG: AT at the boundary between the first intron and the first exon, presumed that the intron cannot be cut out
cuc3-2 proline at position 30 changed to serine by single base substitution
cuc3-3 Tryptophan at position 103 changed to stop codon by 1 base substitution cuc3-4 Glycine at position 25 changed to aspartic acid by 1 base substitution
The nucleotide sequence is determined using an ABI3100 DNA sequencer manufactured by PerkinElmer essentially according to the manufacturer's instructions.
[0079]
Example 4: Assay for transcriptional activation using yeast
Forward primer: 5'-GGAATTCATGCTTGGCGGTGGAAAGATGT-3 '(SEQ ID NO: 6) and
Reverse primer 5'-CCGCCTCGAGCTACAGCTGGAATCCTAAAGG-3 '(SEQ ID NO: 7)
Is used to amplify the CUC3 ORF by PCR using Pyrobest (Takara Shuzo). As a template, cDNA obtained by reverse transcription of total RNA derived from seedlings of Arabidopsis thaliana on the 7th day of germination is used. After cutting the PCR product with EcoRI / XhoI, it is cloned into the EcoRI / XhoI site of pBluescriptIIKS (-). The EcoRI / XhoI fragment is again cloned into the EcoRI / SalI site of pGBT9, transformed into yeast strain Y187 according to yeast protocol handbook of CLONTECH, and β-galactosidase activity is measured. The results indicate that CUC3 functions as a transcription factor.
[0080]
[Table 2]

[0081]
Example 5: Assay of adventitious bud formation ability using transformed callus
Sterile Arabidopsis seeds are sown on a GM medium, and after germination, the hypocotyl is prolonged by low light under a cold gauze. After culturing for 7 days, the hypocotyl is cut out and cultured on CIM medium for 6 days to form a callus. This callus is immersed in B5-MES in which Agrobacterium is suspended to an OD600 of 0.2, and then co-cultured with Agrobacterium on a CIM plate for 2 days. Next, the callus is washed 10 times or more with a washing solution, and then cultured on a SIM selective medium containing an antibacterial antibiotic to induce shoot formation.
The CUC3 cDNA cloned into the above-described pBluescriptIIKS (-) is cut out as a SpeI / XhoI fragment, recloned into the SpeI / XhoI site of pDH123, and transformed into Agrobacterium GV3101 :: pMP90.
[0082]
GM plate
Murashige-Skoog salt 4.3 g / L
Sucrose 10.0 g / L
Myo-inositol 0.1 g / L
0.05% (w / v) MES-KOH (pH 5.7) 5.0 ml / l
Thiamine hydrochloride 20.0 μg / L
Nicotinic acid 1.0 μg / L
Pyridoxine hydrochloride 1.0 μg / L
Biotin 1.0 μg / L
Gellan gum 5.0 g / l
[0083]
Callus induction medium (CIM)
3.3 g / L Gamborg B5 salt
Glucose 20g / L
Myo-inositol 0.1g / L
MES KOH 0.5g / L
Thiamine hydrochloride 20.0 μg / L
Nicotinic acid 1.0 μg / L
Pyridoxine hydrochloride 1.0 μg / L
Biotin 1.0 μg / L
2,4-D 0.5mg / L
Kinetin 0.1mg / L
Gellan gum 7.5 g / L
[0084]
Shoot induction medium (SIM)
3.3 g / L of Gamborg B5 salt
Glucose 20g / L
Myo-inositol 0.1g / L
MES KOH 0.5g / L
Thiamine hydrochloride 20.0 μg / L
Nicotinic acid 1.0 μg / L
Pyridoxine hydrochloride 1.0 μg / L
Biotin 1.0 μg / L
IAA 0.15 mg / L
2iP 0.5 mg / L
Gellan gum 7.5 g / L
[0085]
B5-MES
Gamborg B5 3.3 g / L
MES KOH 0.5 g / L cleaning solution
To B5-MES, 200 mg / L vancomycin and 200 mg / L claforan (cefotaxime sodium) were added.
As a selection medium, SIM was supplemented with 100 mg / L augpenin (potassium clavulanate: ticarcillin sodium = 1: 15) and 20 mg / L hygromycin B.
The results show that transformation with CUC3 cDNA induces adventitious shoots from callus.
[Table 3]

[0086]
【The invention's effect】
The novel genes according to the invention can be used to provide molecular biological means for controlling side branch formation in plants. By this means, a remarkable effect is obtained that it is possible to obtain a plant line having a changed side branch formation characteristic in plant breeding. Further, by stably and easily inducing adventitious buds / adventitious roots from cultured plant cells using the novel gene of the present invention, a remarkable effect that a clone of the same genotype can be mass-proliferated is brought about. It is.
[0087]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows cup shaped cotyledons of cuc1cuc2, cuc1cuc3 and cuc2cuc3 mutants.
FIG. 2-1 shows the nucleotide sequence of CUC3.
FIG. 2-2 shows the nucleotide sequence of CUC3.
FIG. 2-3 shows the nucleotide sequence of CUC3.
FIG. 2-4 shows the nucleotide sequence of CUC3.
FIG. 3 shows two subtype mutants of Arabidopsis thaliana, wild-type and cuc2cuc3. While wild-type normally formed side buds, cuc2-1cuc3-2 inhibited side bud formation and cuc2-1cuc3-1 completely inhibited side bud formation.
FIG. 4 shows the amino acid sequence of CUC3 protein.

Claims (12)

(A) the nucleotide sequence of SEQ ID NO: 1,
(B) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added,
(C) a complementary sequence of (a) or (b), and (d) a nucleotide sequence capable of hybridizing to any of the nucleotide sequences of (a) to (c) under stringent hybridization conditions;
A DNA comprising a nucleotide sequence selected from the group consisting of: (A) positions 1 to 205, 338 to 612, 1283 to 1807, 64 to 205, 338 to 612, 1283 to 1315, 1 to 23, 1786 to 1807 or 1345 to 1364 of SEQ ID NO: 1 A nucleotide sequence indicated by a position,
(B) a nucleotide sequence encoding the amino acid sequence of positions 22 to 171 of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added,
(C) a complementary sequence of (a) or (b), and (d) a nucleotide sequence capable of hybridizing to any of the nucleotide sequences of (a) to (c) under stringent hybridization conditions;
A DNA comprising a nucleotide sequence selected from the group consisting of: A protein comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added. A protein comprising the amino acid sequence at positions 22 to 171 of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted or added. A recombinant DNA construct comprising the DNA of claim 1. A plant cell, tissue, organ, plant or progeny transformed with the recombinant DNA construct of claim 5. A method for substantially increasing or decreasing lateral bud formation of a plant as compared to a control plant, wherein the expression level of the DNA of claim 1 or 2 is substantially increased or decreased as compared to a control plant. The method characterized by the above. The method of claim 7, further comprising transforming a plant with a recombinant DNA construct comprising the DNA of claim 1 or 2 and a promoter operably linked thereto. 8. The method of claim 7, further comprising transforming a plant with a recombinant DNA construct comprising a sequence that reduces expression of the DNA of claim 1 or 2 and a promoter operably linked thereto. A method for forming adventitious buds from cultured plant cells by transforming the cultured plant cells with the recombinant DNA construct of claim 5. A method for producing a plant, comprising transforming a cultured plant cell with the recombinant DNA construct of claim 5 to form an adventitious bud from the cultured plant cell. A plant cell, tissue, organ, plant or progeny obtained by the production method of claim 11.

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