KR102271885B1 - IAA2 mutant for controlling plant root hair growth and uses thereof - Google Patents

Abstract

The present invention provides IAA2 protein variants in which the 17th and 66th amino acids of Arabidopsis thaliana-derived IAA2 (Indole-3-acetic acid inducible 2) protein consisting of the amino acid sequence of SEQ ID NO: 1 are substituted with alanine and serine, respectively, encoding the variant To a gene and its use, the IAA2 protein mutant of the present invention may be usefully used to improve traits related to root hair elongation of plants and to search for genes regulating root hair elongation in other plants.

Description

IAA2 mutant for controlling plant root hair growth and uses thereof

The present invention relates to an IAA2 (Indole-3-acetic acid inducible 2) mutant that regulates root hair elongation of plants and uses thereof.

The roots of a plant not only absorb water and nutrients, but also provide the physical strength to support the above-ground parts. Root hairs are tubular forms that arise from the root epidermis, increasing the surface area of the root hairs by about two times and allowing the roots to reach soil particles or necessary nutrients. Root hairs are considered important for nutrient absorption, and their response to nutrient stress has been studied. In adult plants with developed secondary root structures, the side roots were found to be central to the role of supporting plants and anchoring them to the soil. However, unlike adult plants, seedlings have only early-developed roots and root hairs, so research on the role of root hairs, for example, soil fixation, is needed, but it has not been well studied.

Since most seeds germinate at the soil surface, seedlings growing on the soil surface can easily expose their roots to even a small extent of soil loss. Under these circumstances, it is not known whether the root hairs, which are the only additional organs in the roots of seedlings, are advantageous for the seedlings to survive the soil loss environment without being washed away or to survive in the environment where the roots are exposed above the soil surface.

In the present invention, using five transgenic plant species, only the root hair length phenotype was used to check the soil adhesion and water adsorption capacity of the roots among the seedlings, and thereby the survival rate of the seedlings was confirmed.

On the other hand, Korean Patent Application Laid-Open No. 2011-0006330 discloses 'RHS10, a gene for regulating plant root hair development and a method for regulating plant root hair development using the same,' and Korean Patent Application Publication No. 2012-0004167 discloses 'through auxin transporter expression. A method for controlling root hair elongation of a plant and a plant according thereto are disclosed, but the IAA2 mutant for controlling root hair elongation of a plant of the present invention and use thereof are not described.

The present invention was derived from the above needs, and the present inventors conducted a study on the effect of the root hair length of seedlings on the soil and moisture adsorption and survival of roots, Arabidopsis-derived RHS10 (ROOT HAIR SPECIFIC 10), axr2 -1 (mutation at amino acid position 87 of IAA7 (Indole-3acetic acid inducible 7) protein), RSL4 (ROOT HAIR DEFECTIVE SIX-LIKE 4), IAA2mImⅡ (IAA2 (Indole-3acetic acid inducible 2) protein at amino acid 17th and 66th position) Mutant) As a result of root tissue-specific expression of the protein-coding gene, it was found that RHS10 or axr2-1 overexpressing plants had shorter or no root hairs compared to the control, whereas RSL4 or IAA2mImⅡ overexpressing plants had longer root hairs compared to the control. The present invention was completed by confirming that the longer the root hair is, the easier it is for soil adhesion and moisture adsorption.

In order to solve the above problems, the present invention is Arabidopsis thaliana consisting of the amino acid sequence of SEQ ID NO: 1 IAA2 (Indole-3-acetic acid inducible 2) derived from IAA2 (Indole-3-acetic acid inducible 2) protein 17th and 66th amino acids, respectively, alanine (alanine, A) and IAA2 protein variants substituted with serine (S) are provided.

In addition, the present invention provides a gene encoding the IAA2 protein variant.

In addition, the present invention provides a recombinant vector comprising the gene.

In addition, the present invention provides a host cell transformed with the recombinant vector.

The present invention also provides a method for increasing root hair elongation of a plant, comprising the step of transforming a plant cell with the recombinant vector to overexpress a gene encoding a mutant IAA2 protein.

In addition, the present invention provides a method for producing a transgenic plant having increased root hair elongation by transforming plant cells with the recombinant vector.

In addition, the present invention provides a transgenic plant with increased root hair elongation prepared by the above production method and a seed thereof.

In addition, the present invention provides a composition for enhancing root hair extension of a plant comprising a gene encoding a mutant IAA2 protein as an active ingredient.

The IAA2 protein mutant of the present invention can be usefully used to improve traits related to root hair elongation of plants and to search for genes regulating root hair elongation in other plants. In addition, it is possible to create a plant in which the root hair elongation of the plant is regulated by using the IAA2 protein mutant coding gene of the present invention.

1 shows the length of root hairs of different transgenic plants. (a) Control ( ProE7:YFP ) and RHS10 overexpressing plants (RHS10ox, ProE7:RHS10 ), axr2-1 overexpressing plants (axr2-1ox, ProE7:axr2-1 ), RSL4 overexpressing plants (RSL4ox, ProE7:RSL4 ), and This is a representative photo of the root of an IAA2mImII overexpressing plant (IAA2mImIIox, ProE7:IAA2mImII). Bar = 100 μm (all figures applied) (b) Result of quantitative root hair length. Data represent mean ± standard error (n = 151-511 root hair length measurements from 13-39 roots). (c) Quantification results of the thickness and length of the main root. Data represent mean ± standard error (n = 12-36 root length measurements). P < 0.05, applied to b and c.
Figure 2 shows the seedlings attached to the soil after induction of soil loss by pouring water. (a) A schematic diagram of the entire experimental process, showing the appearance of seedlings remaining without being washed away after inducing soil loss by pouring water into the soil with seedlings (see FIG. 6). (b) The ratios remaining in the soil in the control and transgenic plants ( RHS10ox , axr2-1ox , RSL4ox , IAA2mImIIox) after watering were shown. Data represent mean ± standard error (n = 5). P < 0.05.
Figure 3 shows the soil fixation ability of the roots of seedlings. (a) A representative view of the roots and fixed soil of the control and transgenic plants ( RHS10ox , axr2-1ox , RSL4ox , IAA2mImIIox) after being pulled from the soil. Bar=5 mm (all figures apply). (b) The weight of the soil attached to the uprooted root. Data represent mean ± standard error (n = 11 to 26). P < 0.05. (c) It is an enlarged picture of the soil attached to the root hairs. Bar=0.5 mm (all figures apply).
Figure 4 shows the viability of the seedlings exposed to the roots on the soil surface. (a) Representative views of the control and transgenic plants ( RHS10ox , axr2-1ox , RSL4ox , IAA2mImⅡox ) with exposed roots on the soil surface. Bar=1 cm (all figures apply). (b) The survival rate of each experimental group after 15 days of placing the seedlings on the soil surface. Data represent mean ± standard error (n = 3). P < 0.05.
Figure 5 shows the water adsorption capacity of the seedling roots. (a) The seedlings of the control and transgenic plants ( RHS10ox , axr2-1ox , RSL4ox , IAA2mImIIox ) were extracted from the medium and placed on a glass, and the appearance of the roots and water film immediately after extraction and the root and water film disappeared after the moisture was completely dried This is a representative picture of the appearance. Bar=500 μm (all figures apply). The water adsorption capacity of the roots was applied by quantifying the width of the water film including the roots as shown in the red line shown in the RSL4ox photo. (b) The change in moisture adsorbed to the roots was quantified every 15 seconds after the roots were pulled out of the medium. Data represent mean ± standard error (n = 25 roots in each transgenic plant). The statistical significance of the difference in water film width of each transgenic plant immediately after being pulled out of the medium was marked with different alphabets. P < 0.05.
6 shows a water pouring experiment inducing soil loss. (a) The size of the box and the diameter of the nozzle of the water stopper were marked as the preparation for the water pouring experiment observed above. (b) The water pouring experiment was observed from above. (c) A side view of the water pouring experiment.
7 shows the moisture adsorption trend of the roots according to time. Root photos of seedlings of control and transgenic plants ( RHS10ox , axr2-1ox , RSL4ox , I AA2mImIIox ), starting immediately after the root is removed from the medium, and taking it until the width of the water film does not change any more. The red arrow head indicates the area where the water film area is quantified. Bar=500 μm (all figures apply).

In order to achieve the object of the present invention, the present invention provides that the 17th and 66th amino acids of Arabidopsis thaliana derived IAA2 (Indole-3-acetic acid inducible 2) protein consisting of the amino acid sequence of SEQ ID NO: 1 are alanine ( IAA2 protein variants substituted with alanine, A) and serine (S) are provided.

When the IAA2 protein mutant according to the present invention is overexpressed in a transformed plant, the length of the root hair of the plant can be significantly increased.

The present invention also provides a gene or polynucleotide encoding the IAA2 protein variant. In the following specification, a gene encoding an IAA2 protein variant may be used interchangeably with a polynucleotide encoding an IAA2 protein variant.

The range of the gene encoding the IAA2 protein variant according to the present invention is, in the sequence of the wild-type IAA protein consisting of the amino acid sequence of SEQ ID NO: 1, the 17th amino acid cysteine (cystein, C) is substituted with alanine (alanine, A) and , so that the 66th amino acid, proline (P), can be substituted with serine (S), the coding base for cysteine is the coding base for alanine (GCT, GCC, GCA, or GCG), and the coding base for proline is It may be substituted with a coding base of serine (TCT, TCC, TCA or TCG), but is not limited thereto.

The present invention also provides a recombinant vector comprising a gene encoding the IAA2 protein variant.

The gene encoding the IAA2 protein variant of the present invention may consist of the nucleotide sequence of SEQ ID NO: 5, but is not limited thereto.

The term "recombinant" as used herein refers to a cell in which the cell replicates, expresses a heterologous nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form. Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.

As used herein, the term “vector” is used to refer to a DNA fragment(s), a nucleic acid molecule, that is delivered into a cell. The vector replicates DNA and can be reproduced independently in a host cell. The term "carrier" is often used interchangeably with "vector." The term "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotes are known.

A preferred example of a plant expression vector is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-DNA region, into a plant cell when present in a suitable host such as Agrobacterium tumefaciens. Another type of Ti-plasmid vector (see EP 0116718 B1) is currently used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0120516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors such as those that can be derived from double-stranded plant viruses (eg CaMV) and single-stranded viruses, gemini viruses, etc. For example, it can be selected from incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to adequately transform a plant host.

The expression vector will preferably contain one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate, phosphinothricin and glufosinate, kanamycin, G418, bleomycin, hygromycin, Antibiotic resistance genes such as chloramphenicol, but are not limited thereto.

In the plant expression vector of the present invention, the promoter may be a root hair-specific expression promoter, EXPA7 (expansin A7), EXPB5 (expansin B5), or EXPB1 (expansin B1), but is not limited thereto. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. In the plant expression vector of the present invention, since the selection of transformants can be made by various tissues at various stages, a constitutive promoter in addition to the above promoter can also be used in the present invention. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental states or cell differentiation. The constitutive promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto, and the selectivity is not limited thereto.

In the plant expression vector of the present invention, a conventional terminator may be used, for example, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium) tumefaciens ) Octopine (Octopine) gene terminator and the like, but is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a host cell transformed with the recombinant vector.

When the vector of the present invention is transformed into a eukaryotic cell, as a host cell, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines) and plant cells can be used. The host cell is preferably a plant cell.

In addition, the method of delivering the vector of the present invention into a host cell is a microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, DEAE- dextran treatment method, gene bambadment, etc., the vector is transferred to the host cell can be injected into

The present invention also provides a method for increasing root hair elongation of a plant, comprising the step of transforming a plant cell with the recombinant vector to overexpress a gene encoding a mutant IAA2 protein.

In the method of increasing root hair elongation of a plant according to an embodiment of the present invention, the IAA2 protein variant is the 17th of Arabidopsis thaliana-derived IAA2 (Indole-3-acetic acid inducible 2) protein consisting of the amino acid sequence of SEQ ID NO: 1. and amino acid at position 66 may be substituted with alanine and serine, respectively.

The "gene overexpression" means to allow the gene to be expressed at a level higher than that expressed in a wild-type plant. As a method of introducing the gene into a plant, there is a method of transforming the plant using an expression vector containing the gene under the control of a promoter. In the above, the promoter is not particularly limited as long as it can overexpress the inserted gene in the plant.

Transformation of a plant refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Negrutiu et al., 1987, Plant Mol. Biol. 8:363-373), the electroporation of protoplasts (Shillito et al., 1985, Bio/Technol. 3:1099). -1102), microinjection into plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202:179-185), particle bombardment of various plant elements (DNA or RNA-coated) (Klein et al) ., 1987, Nature 327:70), infection with viruses (EP 0301316 B1), etc. in Agrobacterium tumefaciens mediated gene transfer (incomplete) by infiltration of plants or transformation of mature pollen or vesicles. can be chosen A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EPA 120 516 and US Pat. No. 4,940,838.

A "plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants. "Plant tissue" refers to a tissue of a differentiated or undifferentiated plant, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie, single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture state.

The present invention also

transforming plant cells with the recombinant vector to overexpress a gene encoding a mutant IAA2 protein; and

It provides a method for producing a transgenic plant having increased root hair elongation, comprising the step of re-differentiating the plant from the transformed plant cell.

In the method for producing a transgenic plant with increased root hair extension according to an embodiment of the present invention, the IAA2 protein mutant is Arabidopsis thaliana-derived IAA2 (Indole-3-acetic acid inducible 2) protein comprising the amino acid sequence of SEQ ID NO: 1. The 17th and 66th amino acids may be substituted with alanine and serine, respectively, and the plant transformation method and plant cell used for plant transformation are the same as described above.

Transformed plant cells must be redifferentiated into whole plants. Techniques for the redifferentiation of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.).

The present invention also provides a transgenic plant with increased root hair elongation prepared by the above production method and a transformed seed thereof.

The transgenic plant of the present invention may have a 2.2-fold increase in the length of the root hair compared to the non-transformed plant, thereby having excellent moisture and soil adsorption capacity.

In addition, the transgenic plant according to the present invention may be a dicotyledonous plant or a monocotyledonous plant, preferably a dicotyledonous plant. Examples of dicotyledonous plants include, but are not limited to, Arabidopsis thaliana, eggplant, tobacco, red pepper, tomato, burdock, radish, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, radish, There are watermelon, melon, cucumber pumpkin, gourd, strawberry, soybean, mung bean, kidney bean and pea.

The present invention also provides a composition for enhancing root hair extension of a plant comprising a gene encoding a mutant IAA2 protein as an active ingredient. The composition for enhancing root hair extension of a plant of the present invention is an active ingredient, wherein the 17th and 66th amino acids of the Arabidopsis thaliana-derived IAA2 (Indole-3-acetic acid inducible 2) protein consisting of the amino acid sequence of SEQ ID NO: 1 are alanine and serine, respectively. It contains a gene encoding the IAA2 mutant protein substituted with , and it is possible to increase the length of the root hair of a plant by transforming the gene into a plant.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

Plants and growing conditions

Columbia Ecotype Arabidopsis thaliana ( Arabidopsis thaliana ) was used as a model plant of the present invention. To quantify root traits, seeds were prepared with 1% sucrose, 0.5 g L ^-1 MES (2-(N-morpholino)ethanesulfonic acid), 0.8% agarose (Duchefa Biochemie, Netherlands) and 30 μg mL ^{-1 Planted in 4.3 g·L −1} MS medium (Sigma-Aldrich, USA) containing hygromycin and titrated to pH 5.7 (KOH). ProE7:YFP was used as a control because it can grow in hygromycin medium and does not affect the growth of root hair length. In an experiment to verify the water adsorption capacity of seedling roots, seeds were planted in 0.3% agarose medium. When growing plants in soil as a continuous experiment, Sunshin Mix #5 (Sun Gro Horticulutre) soil was used after filtering it once using a 1 mm mesh. In other experiments, the same soil was used without sieving. All seeds were subjected to dark treatment before germination and stored in a refrigerator at 4°C for 3 days before use. Seeds were germinated at 22°C under long-day conditions (16 hours light/8 hours dark). The light conditions were ~130 μmol·m ^-2 ·s ^-1 , and the fluorescent lamp used was FHF 32SS-EXD (Kumho Electric). Humidity is 60 ~ 65% relative humidity conditions, other conditions are directly described in the experiment.

transgene structure

Root hair was specifically used as a pCAMBIA1300 vector comprising a ProE7 to express the IAA2mImⅡ (Kim et al, 2006, Plant Cell 18:. 2958-2970). Amplified using mega primers to induce mI mutations in TPL-binding domain I of the IAA2 gene ( AT3G23030 ): 5'-TGATCCCGGGGCAATGGCGTACGAGAAAGT-3' (SEQ ID NO: 2) and 5'-GGTAATCCAAGAGCTAGCTCT-3' (SEQ ID NO: 3) ). Additionally, to obtain a gene sequence induced mutagenesis in the TPL-binding domain II of the IAA2 gene, the genome of an IAA2 gain-of-function mutant plant was used as a template and amplified using a mega primer: mI mega primer (SEQ ID NO: 2 and SEQ ID NO: 3) PCR amplification product used) and 5'-CAAACCCGGGTCAGCTTCTCTGGATCATAAGG-3' (SEQ ID NO: 4). The amplification product was inserted into the vector ProE7p13M digested using the Sma I enzyme. Other transgenes used in the experiment were ProE7:YFP , ProE7:RSL4 , ProE7:RHS10 , and ProE7:axr2-1 (Lee and Cho., 2006, Plant Cell 18:1604-1616; Won et al ., 2009, Plant Physiol. 150:1459-1473; Hwang et al ., 2017, Plant Cell 29:39-53), plants were selected by inserting the corresponding transgene into a wild-type background, and second-generation plants were used.

Measurement of root hair cell length

Digital images of seedling roots grown 3-4 days and 10-13 days after germination were taken with a stereoscopic microscope (M20521 FA, Leica) at 12.5x or 40x magnification. For each variable of the root, ImageJ 1.50b software (National Institutes of Health) was used. The length of root hairs was measured by measuring a total of 16 root hairs from both sides of the root and 8 consecutive fully grown root hairs protruding vertically from each side. The thickness of the main root was measured at three points of the root. The length of the epidermal cells in which roots occur was measured in the section where root hairs were fully grown.

Root and root hair surface area quantification

The surface area of roots and root hairs on seedlings and plants with side roots were directly quantified in the present invention (Table 2) or referenced from photographs of existing research data (Table 1). The root and root hairs were considered cylindrical and the surface area was estimated.

The total root hair area of the plant was calculated from the length and diameter of the root hairs and the total number of root hairs. The surface area of the root excluding the root hairs was determined by measuring the total root length, and in the case of plants with side roots, the total length of the main root and side roots was included. The total root surface area was calculated as the sum of the total root hair surface area and the remaining root surface area. Since the density and length of root hairs at the main and side roots are different, when calculating the total root surface area, the surface area of the root hairs at the main root and the surface area of the root hairs at the side roots were obtained separately and then combined. And the ratio of the surface area of root hairs to the total root surface area was calculated. In order to estimate the total number of root hairs, based on the observation that a total of 8 root hair cells are generated with a 95% probability in epidermal cells of Arabidopsis thaliana (Song et al ., 2011, Plant Physiol. 157: 1196-1208), The root hair density was calculated by dividing the number of root hairs by the total length of the root where root hairs occur. This method was adopted to include root hairs that were not visible in direct observation. The number of root hairs in the total root was calculated by multiplying the root hair density by the total length of the root hairs. Root hair diameter thickness, main root diameter thickness, lateral root diameter thickness, main root length, lateral root length, root apical length, and epidermal cell length in root hairs were measured using the observation results of the present invention (Table 2) and data previously studied (Table 2). One).

Soil loss experiment induced by water pouring

Seedlings grown for 4 days in the medium were transferred to a rectangular box (width 44 cm × length 30 cm × height 8 cm) filled with wet soil and grown for two more days. To create a soil loss environment, the box was tilted 30° and water was poured using a water stopper. The water stopper used was a nozzle with a diameter of 11 cm and 138 holes with a diameter of 1 mm. 2 L of water was poured at a height of 1 m at 133 ml·s ^{−1 ( FIG. 6 ).} Seedlings that were not washed away due to soil loss at this time were considered to be alive. Each experiment was repeated using 30 seedlings.

Soil fixation experiment

In order to use plants with similar root lengths in the present invention, the roots of seedlings grown in the medium for 3 days were observed and then transplanted into the soil. After 2 days, the seedlings were removed from the soil and the weight of the whole seedlings attached to the soil was quantified. In order to know only the weight of the fixed soil, the seedlings were washed with water and dried using thin paper, and then the weight of the seedlings was re-measured. The weight of the seedlings removed from the total weight was taken as the weight of the soil fixed to the root, and this was normalized by dividing it by the length of the main root.

Survival experiments of seedlings on the soil surface

The seedlings grown for 5 days in the medium were transferred to the wet soil surface. No water was given for 14 days, and the number of surviving seedlings (shown in green) after supplying water on the 15th day was quantified. Each experiment was repeated using 40 seedlings.

moisture adsorption experiment

In order to confirm the water adsorption capacity of the roots, seedlings grown for 3 days in 0.3% agar medium were extracted, placed on dry glass, and root photos were taken at 15 second intervals. The maximum horizontal length of the water film around the root including the root was quantified until the roots were completely dried and the water film disappeared (FIG. 7). All experiments were independently performed at least three times.

Example 1. Analysis of the proportion of root hairs in the root surface area of adult plants and seedlings

The roots of seedlings are simply composed of short roots and root hairs. Unlike adult plants with side roots, it is thought that root hairs are required for seedling roots to perform physical functions. Therefore, the present inventors found that the ratio of root hairs to the total surface area of roots of adult plants and seedlings. was calculated. As shown in Table 1, basic data on root hair, main root, and side root were obtained from a total of 15 reference materials. In seedlings 3-5 days after germination, root hairs accounted for about 61% of the total root surface area. In older plants with side roots formed 10 to 13 days after germination, root hairs accounted for about 48% of the total root surface area (Table 1). This result shows that root hairs occupy a high proportion of root hairs in the whole root, especially in seedlings, and this is a result that can be expected to play an important role in the case of seedling roots that have not yet developed side roots.

Example 2. Root hair length analysis of transgenic plants expressing different genes specifically for root hairs

In order to confirm the relationship between root hairs, soil fixation, and survival of seedlings, the present inventors prepared five transgenic Arabidopsis thaliana plants. These are the promoters of the Arabidopsis-derived gene EXPANSIN A7 that specifically overexpress the yellow fluorescent protein (YFP, control), RHS10 ( RHS10ox ), axr2-1 ( axr2-1ox ), RSL4 ( RSL4ox ), or IAA2mImII ( IAA2mImIIox ). It is a transgenic plant. A control group is a plant that can grow in the hygromycin antibiotic medium but does not significantly differ from the root hair length of the wild-type plant in the medium. RHS10ox and axr2-1ox shorten the root hair length. In both plants, the root hairs were very short or did not grow, but did not particularly affect the phenotype of the main root (FIG. 1).

Overexpression of plant RSL4ox of the major proteins in root hair growth RSL4 was hold about 20% of root hairs than the control group (Fig. 1). IAA2mImII is a gene with mutations in the TOPLESS binding motif and the auxin binding motif, and encodes a protein that does not respond to auxin signals and is not degraded. When IAA2mImII used in the present invention was overexpressed, the root hairs were approximately twice as long ( FIG. 1 ). However, there was no particular effect on the length or thickness of the main root (Fig. 1c).

Example 3. Analysis of seedling loss rate according to root hair length in soil loss environment

Rainfall can dislodge soil particles (Uddin et al ., 2015, J. Av. Civil Eng. Prac. Res 1:3-10), which is a situation in which freshly germinated, rooted seedlings are uprooted and exposed to the air. can also make In a soil-loss environment, the survival rate of seedlings depends on the ability of the roots to hold onto the soil or the ability of the roots to withstand gravity before re-entering the soil.

In order to depict the rain falling on the soil, the soil box in which the seedlings were planted was tilted by 30° and 2L of water was poured using a water stopper (FIG. 2). The soil fixation rate was calculated by quantifying the number of seedlings remaining without being swept away in the soil loss situation. This was 76% and 64% for seedlings with long root hairs, such as IAA2mImIIox and RSL4ox, respectively. On the other hand, if the root hair is short and axr2-1ox RHS10ox has shown a fixing rate of 41% and 15% respectively (Fig. 2). The present invention shows that root hairs help the survival of seedlings in soil loss situations such as rain.

Example 4. Soil fixation rate according to the length of root hairs

The high soil fixation rate shown by plants with long root hairs is because the roots of seedlings can hold a lot of soil. To confirm this, the present inventors directly quantified the weight of the soil attached to the root after the seedlings were extracted from the soil. Plants with long root hairs were able to attach about 4 to 8 times more soil to the roots than plants with short root hairs (Fig. 3a, b). These results show that root hairs can significantly improve the soil holding capacity of the roots of seedlings. Therefore, the present invention confirmed that root hairs contribute to increasing the survival rate of seedlings by increasing the soil adhesion ability of the roots.

Example 5. Analysis of the survival rate of seedlings according to the length of root hairs in an environment where root hairs are exposed to the soil surface

If the roots are exposed to the air, the roots are easily dried, which can be fatal to the survival of the seedlings over time. The relationship between the length or presence of root hairs and the survival of seedlings whose roots were exposed to air was checked. Since the amount of attached soil varies depending on the length of the root hairs, seedlings grown in the medium were first extracted and placed on the soil. Seedlings were placed on the soil and live individuals were quantified after 15 days had elapsed. In the present invention, about 40% of the plants with long root hairs, IAA2mImIIox and RSL4ox , survived, but axr2-1 and RHS10ox with short root hairs showed a survival rate of about 10% (Fig. 4). This result can be considered to prove that the longer the root hairs of seedlings, the more favorable the survival in the dry condition where the roots are exposed.

Example 6. Analysis of increase in water adsorption capacity of roots by root hairs

In order for the exposed roots of seedlings to survive in a dry environment before they enter the soil, they must be able to absorb moisture around the roots for a long time. The longer it absorbs moisture, the more favorable it will be for survival. In the present invention, it was found that seedlings with long root hairs adsorbed moisture longer than seedlings with short root hairs. Moisture adsorption capacity was checked by placing the seedlings of wet roots extracted from the medium on glass and quantifying the extent to which the size of the film formed with water around the roots decreased ( FIG. 5 ). As a result of comparing the water adsorption capacity immediately after extraction from the medium, the roots of the seedlings with long root hairs had twice the water adsorption power of the roots of the seedlings with short root hairs (FIG. 5b). In addition, plants with long root hairs held moisture for about 375 to 389 seconds, while plants with short root hairs held water for about 141 to 153 seconds.

In the overall results, plants with long root hairs had better soil and moisture adsorption capacity than plants with short root hairs ( FIGS. 3 and 5 ), and thus the survival rate was also high ( FIGS. 2 and 4 ). Therefore, the IAA2mImII protein variant that induces the longest root hairs is expected to be helpful in increasing the survival rate of seedlings in various environments.

<110> Seoul National University R&DB Foundation <120> IAA2 mutant for controlling plant root hair growth and uses it <130> PN19266 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 174 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Ala Tyr Glu Lys Val Asn Glu Leu Asn Leu Lys Asp Thr Glu Leu 1 5 10 15 Cys Leu Gly Leu Pro Gly Arg Thr Glu Glu Ile Lys Glu Glu Gln Glu 20 25 30 Val Ser Cys Val Lys Ser Asn Asn Lys Arg Gln Phe Glu Asp Thr Arg 35 40 45 Glu Glu Glu Glu Ser Thr Pro Thr Lys Thr Gln Ile Val Gly Trp 50 55 60 Pro Pro Val Arg Ser Ser Arg Lys Asn Asn Asn Ser Val Ser Tyr Val 65 70 75 80 Lys Val Ser Met Asp Gly Ala Pro Tyr Leu Arg Lys Ile Asp Leu Lys 85 90 95 Thr Tyr Lys Asn Tyr Pro Glu Leu Leu Lys Ala Leu Glu Asn Met Phe 100 105 110 Lys Val Thr Ile Gly Glu Tyr Cys Glu Arg Glu Gly Tyr Lys Gly Ser 115 120 125 Gly Phe Val Pro Thr Tyr Glu Asp Lys Asp Gly Asp Trp Met Leu Val 130 135 140 Gly Asp Val Pro Trp Asp Met Phe Ser Ser Ser Cys Lys Arg Leu Arg 145 150 155 160 Ile Met Lys Gly Ser Asp Ala Pro Ala Leu Asp Ser Ser Leu 165 170 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgatcccggg gcaatggcgt acgagaaagt 30 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtaatccaa gagctagctc t 21 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 caaacccggg tcagcttctc tggatcataa gg 32 <210> 5 <211> 525 <212> DNA <213> Artificial Sequence <220> <223> IAA2mImII <400> 5 atggcgtacg agaaagtcaa cgagcttaac cttaaggaca cagagctagc tcttggatta 60 cccggaagaa cagagaagat caaagaagaa caagaggttt cttgcgttaa aagtaacaac 120 aagcgtctat ttgaggaaac tcgtgatgaa gaagaatcta cacctcctac caaaactcaa 180 atcgttggtt ggccatcagt gagatcttcc cgtaagaaca acaacagtgt gagctacgtg 240 aaagtgagta tggacggagc tccttacctt cgcaagatcg atctcaagac atacaaaaac 300 taccccgagc ttctcaaagc gttagagaat atgttcaaag tcatgattgg tgaatattgt 360 gagagagaag gatacaaagg atctggattt gtaccaacat acgaagataa agatggtgac 420 tggatgttgg ttggtgatgt tccatgggac atgttctctt cttcttgtaa gagactcaga 480 atcatgaagg gatccgacgc tcctgctcta gactcttcct tatga 525

Claims (9)

The 17th and 66th amino acids of the Arabidopsis thaliana derived IAA2 (Indole-3-acetic acid inducible 2) protein consisting of the amino acid sequence of SEQ ID NO: 1 are alanine (alanine, A) and serine (serine, S), respectively Substituted IAA2 protein variants. A gene encoding the IAA2 protein variant of claim 1. A recombinant vector comprising the gene of claim 2 . A host cell transformed with the recombinant vector of claim 3. A method for increasing root hair elongation of Arabidopsis, comprising the step of transforming Arabidopsis plant cells with the recombinant vector of claim 3 to overexpress a gene encoding an IAA2 protein variant. Transforming Arabidopsis plant cells with the recombinant vector of claim 3 to overexpress the gene encoding the IAA2 protein mutant; and
Re-differentiating Arabidopsis plants from the transformed Arabidopsis plant cells; Method for producing transgenic Arabidopsis thaliana with increased root hair elongation comprising a. Transgenic Arabidopsis thaliana with increased root hair elongation produced by the method of claim 6 . The transformed seed of Arabidopsis according to claim 7. A composition for promoting root hair elongation of Arabidopsis, comprising the gene of claim 2 as an active ingredient.

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