JP2004350553A - Arabidopsis thaliana AN3 gene - Google Patents

Abstract

A method for controlling a lateral size of a leaf is provided.
The present invention provides an AN3 gene that controls the lateral size of a leaf and a method of changing the shape of the leaf by controlling the expression of the gene.
[Selection diagram] None

Description

【図面の簡単な説明】
【図１】突然変異株（ａｎ３−１）と野生型（ＷＴ）の子葉、ロゼット葉、茎生葉を示す図である。
【図２】突然変異株（ａｎ３−１）と野生型（ＷＴ）の第１葉の葉身の長さ、幅、葉柄の長さの比較を示す図である。
【図３】突然変異株（ａｎ３−１）と野生型（ＷＴ）の第１葉の縦方向と横方向の葉肉細胞数（Ａ）と葉肉細胞の面積（Ｂ）の比較を示す図である。
【図４】突然変異株（ａｎ３−１）と野生型（ＷＴ）の葉肉細胞顕微鏡写真を示す図である。右は突然変異株（ａｎ３−１）、左は野生型（ＷＴ）を表す。
【図５】突然変異株（ａｎ３）と野生型（ＷＴ）のＡＮ３ 遺伝子を増幅するＰＣＲ反応の結果を示す図である。矢印はＡＮ３遺伝子（６３３ｂｐ）を示す。
【図６】ａｎ３−２遺伝子座の変異部位の塩基配列（配列番号１の１〜１２０位）を示す図である。下線は欠失部位を示す。
【図７】変異株（ａｎ）、ａｎ３ 変異株（ａｎ３） 及びａｎとａｎ３の２重変異株（ａｎ ａｎ３）の葉器官を示す図である。
【図８】変異株（ａｎ）、ａｎ３ 変異株（ａｎ３） 及びａｎとａｎ３の２重変異株（ａｎ ａｎ３）の葉器官を示す図である。
【図９】細胞分裂角度の計測を示す図である。
【図１０】ＣＹＣＢ１；２：：ＧＵＳレポータ活性の検出を示す図である。右は突然変異株（ａｎ３−１）、左は野生型（ＷＴ）を表す。黒点はＧ２／Ｍ期の細胞を示す。
【図１１】葉の発生に伴う細胞分裂面頻度を示す図である。右の図は発生ステージの異なる葉原基領域内での細胞分裂面を示す。
【図１２】葉の発生に伴う細胞分裂面の変化を示す図である。
【図１３】１枚の葉原基内の各領域における細胞分裂頻度を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an Arabidopsis thaliana AN3 gene, and more particularly to an AN3 gene that controls the lateral size of a leaf and a method of changing the shape of a leaf by controlling the expression of the gene.
[0002]
[Prior art]
Conventionally, in order to modify the morphology of a plant, in principle, the endogenous amount and sensitivity of plant hormones (gibberellin, brassinosteroid, ethylene, etc.) are adjusted to control the cell size. .
The present inventors have already identified the ROTUNDIFOLIA3 (ROT3) gene belonging to the cytochrome P450 family in Arabidopsis thaliana (Gene & Development 12: 2381-2391 (1998)), and controlled the expression of ROT3 to form leaves and flowers. Can be changed (Non-Patent Document 1). This gene ROT3 is involved only in cell elongation in the vertical direction, and in combination with CYP90D1, cell elongation and, in addition, cell division are changed (Japanese Patent Application No. 2002-248910). In this case it works independently of the polarity of the leaves. In general, brassinosteroids have a promoting effect on both elongation and division of cells in leaves (Non-Patent Document 2). When a deficiency in the function of these genes is caused, the cell elongation is reduced vertically, or both cell division and cell elongation are reduced as a whole.
[0003]
Since these modification methods depend on the existing cell number, the control range of the organ size is limited. In addition, the side effect of changing the physiological function is great.
In addition, the leaves and flower organs of a plant form planes having vertical and horizontal polarities. However, a morphological control method utilizing a cell division process depending on the polar axis is not known.
The inventors have found that there is a mutant called an3 which is not due to a wide cell growth reduction (Non-patent Documents 3 and 4), but its gene was not known.
The amino acid sequence of the Arabidopsis thaliana AN3 gene discovered by the present inventors is a homolog in plants of a factor known as a transcription coactivator in humans and the like (Non-Patent Document 5).
[0004]
[Non-patent document 1]
Proc. Natl. Acad. Sci. USA vol. 96 pp. 9433-9437 (1999)
[Non-patent document 2]
Nakaya, M .; et al. , Plant Cel Physiol. 43: 239-244 (2002)
[Non-Patent Document 3]
Instituto Juan March de Estudios Investigations Center for International Meeting on Biology-Workshop on Leaf Development, February 1, 2001; February 1, 2001;
[Non-patent document 4]
Plant Cell Physiol. 43: 372-278 (2002)
[Non-Patent Document 5]
Brett D. et al. , Hum. Mol. Genet. 6, 1559-1564 (1997)
[0005]
[Problems to be solved by the invention]
There is a limit in controlling the size and morphology of leaf and flower organs only by controlling cell elongation. However, if the number of cells in these organs can be controlled, this limitation can be overcome.
An object of the present invention is to provide a means for adjusting the number of cells in a plant organ, which has higher directivity in the horizontal direction in planar cell growth. Therefore, the plant morphology can be more flexibly adjusted by combining with the method of controlling leaf and flower organ size in the vertical and horizontal directions. This means can be used for developing new varieties of ornamental flowers and ornamental plants having a new form.
[0006]
[Means for Solving the Problems]
The present inventors have analyzed the mutants of Arabidopsis thaliana in the lateral direction, and found the gene AN3 that regulates the number of cells in the lateral direction of leaves and flower organs.
This AN3 is a plant homologue of a factor known as a co-activator in humans and the like. However, its function has been analyzed only in a single-cell culture system and has not been analyzed in a plant at all, and it has never been known that it has a function of controlling leaf morphology.
When the function of the AN3 gene is disrupted, the leaves of the plant become thinner without changing the length. This is because the cell division activity of leaf primordia is reduced. In particular, since the influence on the period when the proliferation of cells in the lateral direction of the leaf is active is strong, the lateral width is strongly affected, not the vertical length.
[0007]
That is, the present invention is a gene comprising the nucleotide sequence of (1) or (2).
(1) Nucleotide sequence of SEQ ID NO: 1 (AN3 gene of Arabidopsis thaliana)
(2) As a result of comparing the N-terminal 125 amino acid residues of the amino acid sequence encoded by SEQ ID NO: 1 with a homologue of the AN3 gene of a plant other than Arabidopsis thaliana, the amino acid sequence of the homologue of the AN3 gene of the plant is AN3 The nucleotide sequence of a homologue of the AN3 gene of the plant when it is included in the same clad group as in the above. The present invention may also be a gene comprising the nucleotide sequence of (1) or (2).
(1) Nucleotide sequence of SEQ ID NO: 1 (AN3 gene of Arabidopsis thaliana)
(2) a cell encoding the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence; Nucleotide sequence having a function of regulating the number The present invention also relates to a method for changing the size of leaves of a plant by controlling the expression of this gene.
Furthermore, the present invention is a plant that has been treated to control the expression of this gene.
Expression of this gene can be controlled by disruption of the gene, introduction of the antisense gene of the gene into damaged plants, RNA interference of the gene, or overexpression of the gene.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The amino acid sequence of AN3 is similar to the human homologue (GenBank, AF244972) on the N-terminal side. According to a BLASTP search, the homology at the N-terminal side is 57%. Although the sequence itself is not similar on the C-terminal side, it is similar in that it has many amino acids such as Q, P, and G. The yeast genome does not contain sequences corresponding to homologues of AN3.
[0009]
Arabidopsis has two other genes that are similar to the AN3 gene (named SYT2, SYT3; GenBank, AY102640, AY102464). As in the case of human, they have high homology only at the N-terminal side of AN3 (about 70 residues). On the other hand, when a TBLASTN search is performed using the N-terminal 125 residue of AN3 as a query, ESTs derived from other plant species having homology even at the 70-125 residue portion are hit. Therefore, a molecular phylogenetic tree is created from the sequence up to the N-terminal 125 residue of AN3 and SYT1, SYT2 and AN3 homologues of other plants, and if the sequence of other plants is included in the same clade as AN3, AN3 Can be regarded as a homolog of
In addition, since the C-terminal side is characterized by "similar amino acid composition", the homology of the amino acid sequence in this portion is low as a whole even if compared with similar genes.
[0010]
The gene ROT3 involved in brassinosteroid synthesis, which the inventors have already found, was involved only in cell elongation in the vertical direction (Non-Patent Document 1). On the other hand, in the case of AN3 this time, the number of cells is significantly lower in the horizontal direction than in the vertical direction because the gene is required when cells actively divide not only vertically but also horizontally in leaves. . However, the size of the cells is larger than the normal type as a result of the leaf-specific compensation.
[0011]
【The invention's effect】
By producing an AN3 antisense gene or RNAi and introducing it into a plant, the number of leaf cells can be controlled and the width can be controlled. That is, expression can be constitutively or organ-specifically induced using the AN3 gene (whole or part of genomic DNA and cDNA) and other plant AN3 homologous genes. In the case of the function-deficient type (antisense method, RNAi method), a decrease in organ size and growth in the width direction can be suppressed.
Furthermore, according to the method of the present invention, plant morphology control can be refined and speeded up as compared with conventional breeding, and development of new varieties can be facilitated.
[0012]
On the other hand, an and an3 work independently, and rot3 and cyp90d1 work independently. Therefore, it is considered that the combination with these control systems for cell size and shape enables free control by a combination of finer or thinner and shorter. Further, as a difference in appearance, the color is different between the thinning (an3) in which the number of cells is reduced and the thinning (an) caused by the small size of the cells. The former has the same color as the normal one, but the latter is darker. This reflects the difference in cell density. This is important for biodesign, especially in flower gardening.
[0013]
Further, by transforming plants using the Arabidopsis thaliana AN3 gene or AN3 homologous gene of other plants, and regulating the cell proliferation ability per leaf and the transformed flower organ of the leaf, the morphology and organ size of the leaf and flower organs are improved. The raw weight can be modified.
[0014]
【Example】
Hereinafter, the present invention is illustrated by examples, but is not intended to limit the present invention.
Example 1
In this example, the number of cells and the width of a leaf organ in the mutant strain (an3) were measured.
The width and length of the leaf organs and the area of the mesophyll cells were determined as follows. First, image data of leaves cut from a plant is collected using a microscope. In the case of observation at the cell level, a leaf organ was subjected to a clearing treatment using a saturated chloral solution (200 g of saturated chloral, 20 g of glycerol, and 50 ml of deionized water). Next, based on the image, the width / length of the leaf organ and the cell area were measured using Image J (http://rsb.info.nih.gov/ij/) of image analysis software.
The number of cells in the width and length directions of the leaves was visually observed by observing the transparent organs under a microscope.
[0015]
FIG. 1 shows a photograph of the leaf organ width in the mutant strain (an3-1) deficient in the Arabidopsis thaliana AN3 gene. The an3 alleles include an3-1 to an3-4, which all have the phenotype shown in FIG. 1 in common. The wild type (WT) is also shown for comparison.
FIG. 2 shows the length of the leaf blade and petiole of the mutant strain (an3-1). The width of the blade blade was almost the same as that of an3-1 and WT, but decreased from 4.6 mm in WT to 3.2 mm in an3-1. Therefore, it can be seen that the mutant strain (an3) has a specifically reduced leaf organ width as compared to the wild type (WT).
FIG. 3 shows the cell number and cell size of the mutant strain (an3-1). In the an3-1 function-deficient mutant strain, the number of cells is reduced (about 60% in the vertical direction and about 50% in the horizontal direction) while the cell size is increased about twice as compared with the wild type. Understand. A photograph of the cells is shown in FIG.
[0016]
Example 2
The mutant strain (an3) was subjected to PCR on the wild-type (WT) genomic DNA using the primers shown in SEQ ID NOs: 2 and 3, and the results are shown in FIG. As a result, it was confirmed that an3-1, an3-3, and an3-4 were deletion mutations of the AN3 gene.
When the nucleotide sequence of the an3-2 gene (SEQ ID NOS: 4 and 5) was determined, a deletion of 6 bases occurred as shown in FIG.
From these results and the result of Example 1, it can be seen that the leaf organ width is controlled by the AN3 gene.
[0017]
Example 3
In this example, the leaf organ width was measured in the Arabidopsis angustifolia mutant (an) and the double mutant of an3 and an (an an3). The results are shown in FIGS. For comparison, the an3 mutant (an3) is also shown.
The AN gene is a gene having homology to the C-terminal Binding Protein of animals (Kim GT et al., EMBO J. 21: 1267-1279 (2002)). In this mutant, the cells constituting the leaf do not grow laterally, and the thickness of the cells constituting the leaf does not grow in the lateral direction. As a result, the leaves become thicker and thinner (Tsuge, T. et al., Development 122: 1589-1600 (1996)).
That is, AN is a control system for cell elongation, and AN3 is a control system for cell division, and the control systems are different from each other. The leaf organ width was further reduced in both double mutants, which also indicates that an and an3 work independently.
[0018]
Example 4
In this example, the nature of cell division during the leaf primordium development process in an3 and WT was analyzed using the promoter :: reporter line of the gene (CYCB1; 2) expressed in the G2 / M phase of cell division. As schematically shown in FIG. 9, by using this reporter line, cell nuclei immediately after cell division and chromosomes during division can be observed by GUS staining (Donnelly, BM et al., Dev. Biol. 215). : 407-419).
FIG. 10 shows an example in which reporter activity in an3 and WT background was detected by GUS staining. Such image data was collected by microscopic observation, and from the stained images of cells during or immediately after division, data in which the cell division surface was written on the image was created. Then, the angles of the leaves of the dividing plane with respect to the vertical axis were tabulated.
[0019]
Within the leaf primordium, a gradient of developmental stage is formed in the vertical direction, and cells that have developed at the tip are included. In FIG. 11, the cell division planes of the young leaf primordium (Early stage) and the more developed leaf primordium (Latte stage) are vertical (0-30 °, 150-180 °), horizontal (60-120 °), and oblique ( 30-60 °, 120-150 °).
In order to examine the change in the cell division surface with the progress of leaf development, the base side (E2) of the early stage and the tip side (E1) and the intermediate part (L2) of the more developed Late stage were compared (FIG. 12). From the results shown in FIG. 11, it was found that the leaf cells contained in the young region at the development stage mainly proliferated in the vertical direction, and then, when the development proceeded, the cells were divided both vertically and horizontally. It was also found that the direction of cell division was not affected by the an3 mutation.
[0020]
From the results of Example 1, it is conceivable that the cell growth in the an3 mutant strain was more affected in the lateral direction than in the vertical direction. Thus, the cell division activity in the leaf primordium of Late stage, in which the cell proliferation in the lateral direction was more active, was examined. With the method used in this example (FIG. 11), the number of dividing cells was counted in each of L1, L2, and L3 regions.
As a result, as shown in FIG. 13, it can be seen that the cell division activity of the basal leaf base is equivalent to that of the wild type strain in the an3 mutant strain, whereas the cell division activity is significantly reduced in L2 and L3. Therefore, it was found that when the function of the AN3 gene was deficient, the stage of active cell growth in the lateral direction in the leaf primordium was shortened, greatly affecting leaf morphology.
[0021]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing cotyledons, rosette leaves, and raw stem leaves of a mutant strain (an3-1) and a wild type (WT).
FIG. 2 is a diagram showing a comparison of the length, width, and petiole length of the first leaf of a mutant strain (an3-1) and a wild type (WT).
FIG. 3 is a graph showing a comparison of the number of mesophyll cells (A) and the area of mesophyll cells (B) in the longitudinal and lateral directions of the first leaf of the mutant strain (an3-1) and the wild type (WT). .
FIG. 4 shows mesophyll micrographs of mutant (an3-1) and wild type (WT) mesophyll cells. The right shows a mutant strain (an3-1), and the left shows a wild type (WT).
FIG. 5 is a view showing the results of a PCR reaction for amplifying the mutant (an3) and wild-type (WT) AN3 genes. The arrow indicates the AN3 gene (633 bp).
FIG. 6 is a view showing the nucleotide sequence of the mutation site at the an3-2 locus (positions 1 to 120 in SEQ ID NO: 1). Underlines indicate deletion sites.
FIG. 7 shows leaf organs of mutant (an), an3 mutant (an3), and a double mutant of an and an3 (an an3).
FIG. 8 shows leaf organs of a mutant (an), an3 mutant (an3), and a double mutant of an and an3 (an an3).
FIG. 9 is a view showing measurement of a cell division angle.
FIG. 10 shows detection of CYCB1; 2 :: GUS reporter activity. The right shows a mutant strain (an3-1), and the left shows a wild type (WT). Black dots indicate cells in G2 / M phase.
FIG. 11 is a diagram showing a cell division surface frequency associated with leaf development. The figure on the right shows the cell division surface in the leaf primordium region at different developmental stages.
FIG. 12 is a view showing a change in a cell division surface accompanying leaf development.
FIG. 13 is a view showing the cell division frequency in each region within one leaf primordium.

Claims (5)

A gene comprising the nucleotide sequence of (1) or (2).
(1) The nucleotide sequence of SEQ ID NO: 1 (2) As a result of comparing the N-terminal 125 amino acid residues of the amino acid sequence encoded by SEQ ID NO: 1 with homologues of the AN3 gene of plants other than Arabidopsis thaliana, When the amino acid sequence of the homologue of the AN3 gene is included in the same clad group as that of AN3, the nucleotide sequence of the homologue of the AN3 gene of the plant A method for changing the size of leaves of a plant by controlling the expression of the gene according to claim 1. The method according to claim 2, wherein controlling the expression of the gene is caused by disruption of the gene, introduction of an antisense gene of the gene into a damaged plant, RNA interference of the gene, or overexpression of the gene. The method described in. A plant that has been treated to control the expression of the gene of claim 1. 5. The method according to claim 4, wherein controlling the expression of the gene is caused by disruption of the gene, introduction of an antisense gene of the gene into a damaged plant, RNA interference of the gene, or overexpression of the gene. A plant according to claim 1.

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