KR101077938B1 - Method for regulating cuticular wax load amounts in plant cell wall and the plant thereof - Google Patents

Abstract

The present invention relates to a method for controlling the loading of cuticle wax on the plant cell wall, and a plant according thereto, and more particularly, a function in which T-DNA is inserted into Arabidopsis-derived AtLTPG1 (Arabidopsis thaliana Lipid Transfer Proteins GPI-anchored) gene. Lost mutant plants; Inserting T-DNA into the Arabidopsis AtLTPG-1 gene to produce a malfunctioning mutant plant, thereby controlling the loading of cuticle wax in the cell wall; GPI (glycosylphosphatidylinositol) -encapsulated lipid transfer protein coding gene AtLTPG1 from Arabidopsis consisting of the nucleotide sequence of SEQ ID NO: 1; And it relates to a composition for controlling the loading of cuticle wax of the cell wall comprising a glycosylphosphatidylinositol (GPI) -encoded lipid transfer protein coding gene derived from Arabidopsis.

Cuticle, Wax, GPI-Anchor, AtLTPG1, Gene, Plant, Composition, Mutation

Description

Method for regulating cuticle wax loading of plant cell wall and plant according thereto

The present invention relates to a method for controlling the loading of cuticle wax on plant cell walls and to plants accordingly.

Plants are exposed to various environmental stresses during growth and development, including droughts, colds, sun exposure and pathogen invasion. The first barrier between vegetation and environmental stress is the cuticle, which acts as a mechanical protection of the organism and prevents the release of moisture to regulate the ingress of foreign matter. In plants, aquatic plants do not develop cuticles but are well developed in terrestrial plants. The cuticle of a plant is a membrane layer of cutin, which is a lead-like or fatty substance covered on the surface of the body. These substances are produced in epidermal cells or tissues inside them, infiltrate the cellulose or pectin layers that form the cell walls, and then secrete and accumulate outside the epidermis. It is well stained with lipid affinity dyes, and is located outside the epidermal outer wall pectin layer and is a layer that can be separated using an organic solvent. Cuticles are in front of leaves, stems, flowers, and fruits. The mature organs develop and become thicker. In addition to the outer cuticle covering the body surface, there is a thin film of lead-like material or cutin on the wall of the cells in contact with the air in the tissues of the leaves and the epidermis. These two cuticle layers are connected to each other via the pore cell surface.

The cuticle is a lipophilic cutin polymer matrix and wax (Holloway PJ (1982) In The Plant Cuticle. Academic Press, London, pp 132). Very long chain fatty acids (VLCFAs, C20 to C34) and their derivatives, cuticle wax, are sandwiched between the cutin polymer substrates. Biosynthesis of cuticle wax is known to occur only in epidermal cells (Suh et al., (2005) Plant Physiol 139: 16491665). During this process, C16 and C18 fatty acids synthesized in the chromosomes are sent to the cytosol, and from the endoplasmic reticulum (ER) to the VLCFAs in the 20 to 34 carbon range by the fatty acid elongase complex. (Millar AA, Kunst L (1997) Plant j 12: 121-131). Extended VLCFAs are modified through two major wax biosynthetic pathways.

Lipid transfer proteins (LTP) have been shown to function to facilitate the transfer of phospholipids from the inside and outside of the plasma membrane (Kader JC (1996) Annu Rev Plant Physiol Plant Mol Biol 47: 627-654). Plant LTPs are small (7-10 kDa) large amounts of basic protein with hydrophobic pockets to facilitate handling of fatty acid or lysophospholipid molecules (Shin et al., (1995) Structure 3: 189-199). The performance of the plant genome and the EST project has revealed the existence of multiple LTP isoforms, which include a variety of functions, including the function of curtin and wax assembly, pathogen protection, cryopreservation, long-distance signal transduction, and cell wall loosening. Presumably related to function (Roy-Barman et al., (2006) Transgenic Res 15: 435-446). Most plant LTPs with curtin monomers and wax transfer function are present in the cell wall (Pyee J, Kolattukudy PE (1995) Plant J 7: 49-59). Accumulation of cuticle wax and increased expression of LTP in tobacco ( Nicotiana glauca ) leaves in drought stressed conditions suggest that LTP is associated with the accumulation of wax (Pyee J, Kolattukudy PE (1995) Plant J 7: 49- 59).

Recently, proteomic analysis revealed 26 Arabidopsis LTP-like proteins comprising a glycosylphosphatidylinositol (GPI) -anchor domain (Borner GHH, Lilley KS, Stevens TJ, Dupree P (2003) Plant Physiol 132 : 568577). However, the function of these proteins is still under study. GPI-anchored proteins (GAPs) are known to attach to the outer surface of the plasma membrane via GPI anchors. In various organisms, including protozoa and vertebrates, the GPI anchor attachment process involves cleavage of the C-terminal carboxyl group of proteins in ER-lumens and ethanolamine phosphate, trimannoside, glucosamine and inositol phospholipids. A minor addition of the core structure that was made (Udenfriend S, Kodukula K (1995) Annu Rev Biochem 64: 563591). GPI-anchors are involved in plasma membrane targeting, polarized targeting of apical plasma membranes, lipid rafts association, plasma membrane regeneration, signal detection and transformation (Mayor S, Riezman H (2004) Nat Rev Mol Cell Biol 5: 110-120). At present, there has been no report on the effects on plants when glycosylphosphatidylinositol (GPI) -encoded lipid transfer protein coding gene (AtLTPG1) derived from Arabidopsis has been dysfunctional as in the present invention.

The present invention has been made in accordance with the above requirements, the present invention is the total amount of wax of the stem, leaves and long cell wall of the malfunctioning mutant plants produced by inserting T-DNA into the AtLTPG1 gene derived from Arabidopsis larvae wild type ( On the basis of the reduced facts, the present invention aims to provide a method for controlling the loading of cuticle wax on plant cell walls.

In order to solve the above problems, the present invention provides a malfunctioning mutant plant in which T-DNA is inserted into the Arabidopsis thaliana Lipid Transfer Proteins GPI-anchored (AtLTPG1) gene.

The present invention also provides a method of controlling the loading of the cuticle wax of the cell wall, comprising inserting T-DNA into the Arabidopsis-derived AtLTPG1 gene to prepare a malfunctioning mutant plant.

The present invention also provides a glycosylphosphatidylinositol (GPI) -encapsulated lipid transfer protein coding gene AtLTPG1 derived from Arabidopsis, consisting of the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a composition for controlling the loading of the cuticle wax of the cell wall, which comprises a glycosylphosphatidylinositol (GPI) -encoded lipid transfer protein coding gene derived from Arabidopsis.

The method of controlling the loading amount of the cuticle wax of the plant cell wall developed in the present invention can provide information that the plant can effectively defend against invasion of pathogens including fungi.

In order to achieve the object of the present invention, the present invention provides a malfunctioning mutant plant in which T-DNA is inserted into Arabidopsis thaliana Lipid Transfer Proteins GPI-anchored (AtLTPG1) gene. The mutant plant developed in the present invention is a mutant in which T-DNA is inserted into the second exon of the AtLTPG1 gene and the function of the AtLTPG1 gene is lost. The gene may be composed of the nucleotide sequence of SEQ ID NO: 1, but variants of the nucleotide sequence are included within the scope of the present invention.

The plant according to an embodiment of the present invention is Arabidopsis, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gatmu, It may be a dicotyledonous plant characterized in that it is selected from watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean and pea, but is preferably Arabidopsis.

The present invention also provides a method of controlling the loading of cuticle wax in the cell wall, comprising inserting T-DNA into the Arabidopsis-derived AtLTPG1 gene to prepare a malfunctioning mutant plant. As a method for preparing a malfunctioning mutant by inserting T-DNA into the AtLTPG1 gene, a general method known in the art may be used.

Total wax loading of stem, leaf and long cell walls of the atltpg1 mutant was reduced by 5 to 32% compared to wild-type (Col-O), and C29 alkanes (nonacecine), a major component of stem and long cuticle waxes ) Decreased to 30-50% and C28 fatty acids and C24 ~ C28 primary alcohols decreased 12-12% in stems, leaves and long shells.

In a method according to an embodiment of the present invention, the gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.

In the method according to an embodiment of the present invention, the plant may be a dicotyledonous plant, but is preferably Arabidopsis.

In the method according to an embodiment of the present invention, the plant is characterized by an increased sensitivity to fungi. The fungus is preferably Alternaria brassicicola , but is not limited thereto. The wild type (Col-0) infected with Alternaria Brassicchia coli formed a small brown necrotic injury site, but the atltpg1 plant formed a widespread injury site by infection with lactophenol -aniline blue staining.

The present invention provides a GPI (glycosylphosphatidylinositol) -encapsulated lipid transfer protein coding gene AtLTPG1 derived from Arabidopsis, consisting of the nucleotide sequence of SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1 . The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a composition for controlling the loading of the cuticle wax of the cell wall containing a GPI (glycosylphosphatidylinositol) -encoded lipid transfer protein coding gene derived from Arabidopsis.

In the composition of the present invention, the GPI (glycosylphosphatidylinositol) -encoded lipid transfer protein coding gene may be preferably composed of the nucleotide sequence represented by SEQ ID NO: 1. The composition for controlling the loading amount of cuticle wax of the plant cell wall of the present invention contains the AtLTPG1 gene derived from Arabidopsis as an active ingredient, and reduces the loading amount of the cuticle wax of the plant cell wall by losing the gene AtLTPG1 in the plant, thus containing the gene. One composition can be used to control the amount of cuticle wax in the cell wall.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these examples.

Materials and methods

1. Plants

Arabidopsis (Arabidopsis thaliana eco-type Columbia-0) was grown in Arabidopsis growth chambers on a 16h-myeong / 8h-cancer cycle. Seeds were used after soaking in 70% (v / v) ethanol for 1 minute, sterilized in 20% (v / v) lacx for 5 minutes, rinsed with sterile water. This sterilized seed was germinated sterile in 0.8% (w / v) 1/2 MS agar medium with 1% (w / v) sucrose. To treat abiotic stress induction and the stress hormone ABA, 10-day seedlings were added to MS liquid medium with 1 M ABA, 20% (w / v) polyethylene glycol (PEG), 200 mM NaCl, or 200 mM mannitol. Incubated for 6 hours at.

2. Genetic Analysis

Petals, calyx, and stamens in unopened buds were castrated and crossed using pistils as pollen recipients. Homozygous atltpg1 mutants were reciprocally crossed with wild type plants (Col-0). After harvesting, the hybridized seeds were grown until the harvest of F ₂ seeds. The atltpg1 mutant is resistant to phosphinothricin, so genetic separation of F ₂ is achieved by adding 4 μg mL ^-1 phosphinothricin (w / v). Seeds were germinated in 1/2 MS agar medium.

3. RNA Isolation and RT-PCR Analysis

Total RNA was isolated from various Arabidopsis tissues using TRIzol reagent (Sigma, USA) according to the manufacturer's instructions. Reverse transcription was performed as directed by the manufacturer (Invitrogen, USA). PCR was performed using the gene-specific primers shown in Table 1.

TABLE 1 Primers used in the present invention (numbers in parentheses indicate SEQ ID NO.)

4. Construction of Binary Vectors and Arabidopsis Transformation

For isolation of 5'-flanking regions of the AtLTPG1 gene, chromosomal DNA was extracted from 10-day Arabidopsis seedlings by Weigel and Glazebrook (Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory manual, Ed 3 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The 5'- flanking region of the AtLTPG1 gene, approximately 2.2 kb, was amplified by PCR using AtLTPG-1 GUS F1 / AtLTPG-1 GUS R1 primers. The 5'-flanking region of the isolated AtLTPG1 gene was cleaved with Xba I / Sma I restriction enzyme and linked to pBI101 vector cleaved with Xba I / Sma I (Jefferson et al., (1987). EMBO J 6: 3909-3907). to perform a test of complementarity atltpg-1 mutants, AtLTPG1 genomic DNA coding and non-coding region of the promoter, AtLTPG1 genes (approximately 3.8 kb) was amplified by PCR using the GUS AtLTPG1 F1 / R3 primer AtLTPG1 I was. XbaI / SmaI-cleaved genomic DNA fragments were cloned into Xba I / Sma I-cleaved pBIN19 vectors (Bevan MW (1984) Nucl Acids Res 12: 8711-8721). Agrobacterium strain GV3101 was transformed using the prepared binary vector by a freeze thaw method (An G (1987) Meth Enzym 153: 292-305), and the Arabidopsis wild type (Col-0) was converted to Bechtold et al. (Bechtold et al., (1993 ) CR Acad Sci Paris Life Sci 316: 1194-199) was transformed using the vacuum infiltration method. Seeds harvested from each pot were germinated and then germinated in 1/2 MS agar medium with 50 μg mL ⁻¹ kanamycin (w / v) added. The surviving T ₁ or T ₂ seedlings were transferred to the soil and continued for analysis.

5. Histochemical Analysis of β-glucuronidase (GUS)

Histochemical analysis of GUS activity was performed according to the method of Jefferson et al. (Jefferson et al., (1987) EMBO J 6: 3909-3907). In brief, developed long shells, leaves, caulines, flowers, stems and roots were cut with a sharp razor and then GUS staining buffer [100 mM sodium phosphate, pH 7.0, 1 mM 5-bromo-4-chloro-3 -Indolyl-β-D-glucuronide, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferrocyanide, 10 mM Na ₂ EDTA and 0.1% (v / v) Triton X-100] at 37 ° C. for 16 hours. Dyeing reaction. The stained tissue was rinsed with a series of graded ethanol until the pigment, such as chlorophyll, was completely cleared. Next, images were taken with a LEICA L2 microscope (LEICA, Germany). To observe the cross-sections of the stems and leaves, the dehydrated samples were inserted into acrylic resin (LR White Resin; London Resin Company) and then 2 mm sections using ultra-microtome (RMC MT X, USA). Cut to Tissue sections were observed under light microscopy (LEICA, L2, Germany).

6. Microstructure Analysis Using TEM

Arabidopsis wild-type and atltpg 1 mutant stems and leaves were fixed for 4 hours at 4 ° C. with a solution containing 2.5% glutaraldehyde and 4% para-formaldehyde in 0.1 M phosphate buffer. Rinse with 0.1 M phosphate buffer (pH 7.4) and then fix with 4% (w / v) osmium tetraoxide (OsO ₄ ) at 4 ° C. Samples rinsed with 0.1 M phosphate buffer were dehydrated and placed in LR White Resin (London Resin Company). Thin sections (50-60-nm thick) were prepared with ultra-microtome (RMC MT X, USA) and collected on nickel grids (1-GN, 150 mesh). These sections were then stained with uranyl acetate and red citrate and observed by transmission electron microscopy (Philips, Tecnai 12, The Netherlands).

7. Nile Red Dyeing

Epidermal shells of Arabidopsis wild-type and atltpg1 mutant stems were reacted for 5 minutes with a 75% (v / v) glycerol solution containing 25 μg mL ⁻¹ Nile Red, washed with sterile water and confocal microscopy (OLYMPUS, IX71). , Japan).

8. Wax Analysis

The skin wax was extracted by soaking 10 cm flowering stems or leaves in 5 mL chloroform for 30 seconds at room temperature. n - octanoyl Kosan (n -Octacosane), Toko Inc. acid (docosanoic acid) and 1-tree play Kosa (1-tricosanol) was used as the internal standard (internal standards). The solvent was heated to 40 ° C. with a gentle stream of nitrogen to remove residual nitrogen. Wax mixture remaining in the service with pyridine (20 minutes, 100 ℃) to deform in trimethyl silnil derivative corresponding to all the compounds containing hydroxyl groups - N, N - (trimethylsilyl) acetamide trifluoroacetate (bis- N , N- (trimethylsilyl) trifluoroacetamide, BSTFA, Sigma) were treated. The qualitative composition of the mixture is capillary GC-MS (GCMS-QP2010, Shimazu, Japan; column 60 m HP-5, 0.32-mm id, df = 0.25) with He carrier gas at 1.0 mL min ^-1 injection pressure m, Agilent, USA) and mass spectrometry detectors (GCMS-QP2010, Shimazu, Japan). The GC-MS protocol is as follows: injection at 220 ° C., hold at 220 ° C. for 4.5 minutes, increase to 290 ° C. at 3 ° C. min ⁻¹ rate. The temperature was then maintained at 290 ° C. for 10 minutes and increased to 300 ° C. at a rate of 2 ° C. min ⁻¹ for 10 minutes. Quantitative analysis of the mixture was performed using capillary GC as a flame ionization detector under the same conditions as the GC conditions described above. Single compounds were quantified for internal standards by automatically incorporating peak regions.

9. Fungal Pathogen Treatment

Inoculation of Alternaria brassica cola was performed by dropping 10 μL of spore suspension (1 × 10 ⁶ spores mL ⁻¹ ) onto the leaves of each of the 4 week plants. Damaged sites and fungal hyphae were analyzed microscopically by staining the infected leaves by the method described above (Kumar et al., (2004) J Biosci 29: 23-31). Leaves were detached 3 days after inoculation and stained with lactophenol-aniline blue. The lactophenol was prepared by mixing with phenol: acetic acid: sterile water: glycerol (1: 1: 1: 2, v / v / v / v). Aniline blue (0.1%, w / v) and lactofetol were mixed at 1: 3 (Koch and Slusarenko, 1990). Stained samples were prepared in 60% glycerol and examined by light microscopy (LEICA, L2, Germany).

10. EYFP Tagged (Enhanced Yellow Fluorescence Protein-Tagging) and Intracellular Location of AtLTPG-1

EYFP was amplified by PCR using the p35S-EYFP-Flag / Strep plasmid as a template using the YFP-F1 / YFP-R1 primer. Total length AtLTPG1 cDNA used AtLTPG-1 cDNA / AtLTPG-1 P1 and AtLTPG-1 P2 / AtLTPG-1 cDNA primers respectively to amplify AtLTPG-1 cDNA1 fragments and AtLTPG-1 cDNA2 fragments. Tian et al., (2004) Plant Physiol for making AtLTPG1 cDNA with EYFP 135: 25-38, a triple template PCR (TT-PCR) was performed according to the method described. The TT-PCR product was digested with Sma I / BamH I enzyme and linked to p35S-EYFP-Flag / Strep plasmid digested with Sma I / BamH I.

The constructed plasmid was purified using Qiagen Plasmid Maxi kit (Hilden, Germany). Arabidopsis foliar protoplasts were prepared by slightly modifying the method described above (Abel S, Theologis A (1998) Methods Mol Biol 82: 209-217). Briefly, rosette leaves of 2-3 weeks old Arabidopsis plants grown with 16h-myeong / 8h-cancer photoperiod were collected and cut, and cut solutions (1% cellulase R10, 0.25% maserozyme R-10, 400 mM mannitol). , 8 mM CaCl ₂ , 5 mM Mes-KOH, pH 5.6) for 3 hours, at 40 ° C. at room temperature was added to light agitation with agitation. Protoplasts were collected through a sieve (100 μm mesh), loaded with 0.45 M sucrose and centrifuged at 200 × g for 5 minutes. The border of the green-band was removed and 10-20 mL of W5 solution (154 mM NaCl, 125 mM CaCl ₂ , 5 mM KCl, 5 mM glucose, 1.5 mM Mes-KOH, pH 5.6) was added, and 200xg for 5 minutes. Centrifuged. The pellet was mixed with 10 mL of W5 solution and kept on ice for 30 minutes. After centrifugation, the protoplasts were mixed with MaMg solution (400 mM mannitol, 15 mM MgCl 2, 5 mM Mes-KOH, pH 5.6) and then infected with plasmid DNA purified by polyethylene glycol (PEG) -mediated protoplast transformation. Infected protoplasts were reacted for 18 hours in dark at room temperature, and fluorescence was observed using Nikon ECLIPSE TE2000-U, Japan.

Example 1 Encoding GPI-anchored Lipid Transfer Protein AtLTPG1  Isolation of Genes>

Suh et al. (2005) Plant Physiol 139: 1649-1665 reported that the transcriptome of Arabidopsis stem and stem epidermal cells was analyzed to identify genes associated with surface lipid metabolism. It was. This analysis identified 7 LTP candidate proteins that may be associated with wax and / or cutin monomer delivery. Among these proteins, AtLTPG1 (At1g27950), which is expressed more in epidermal cells of stems than in stems, was selected on the basis of specific LTP structures, including long C-terminal regions, which have yet to be well known.

In addition, the similarity (%) between the AtLTPG-1 gene and the genes present in other plants is shown in FIG. 9. In the case of Alpine phencrere ( Thlaspi caerulescens ), 81% confirmed that the ABI81469 gene having the highest similarity with AtLTPG-1 exists. Lichens ( P. patens, Physcomitrella patens subsp ), Sitka (wood) ( Picea sitchensis ), Grapes ( Vitis vinifera ), Rice ( Oryza sativa ), Arabidopsis thaliana , Alpine penicres ( Thlaspi caerulescens ), Chickpeas ( Genes found to be highly homologous to AtLTPG-1 in Cicer arietinum ) and maize ( Zea mays ) were found to contain conserved cysteine residues as well as cysteine residues present in AtLTPG-1. This is expected to be similar.

To confirm the expression level of AtLTPG1 transcripts in the stem and epidermal husks, total RNA was isolated and RT-PCR was performed. Arabidopsis actin2 gene (At3g18780) was used for qualitative and quantitative cDNAs. Similar to the microarray results, the expression level of AtLTPG1 transcript was about 2 times higher in the stem epidermal shell than in the stem (FIG. 1A).

Isolation of AtLTPG1 genomic DNA (approximately 3.8 kb) from young seedlings grown on day 10 revealed that genomic DNA contained two exons and one intron containing GT-AG nucleotides at both ends (data not shown). AtLTPG1 protein was found to contain three different domains. Specifically, the 22 amino acid residues at the N-terminus were presenting the signal peptide required for protein secretion. In addition, amino acid residues present between 35 and 116 residues that exhibit approximately 60% homology with the nonspecific LTP may have eight highly conserved cysteine residues at positions 35, 45, 60, 61, 74, 76, 106, and 116. I had. Finally, the C-terminal end (from residue 168 to 191) included the sequence encoding the GPI-anchor domain (FIGS. 1B and C).

<Example 2. AtLTPG1  Expression site and time of expression of transcript>

To investigate the expression of AtLTPG1 transcripts, total RNA was extracted from roots, seedlings, leaves, caulines, flowers and siliques and RT-PCR analysis was performed. AtLTPG1 transcript was expressed in all tissues examined (data not shown). To investigate whether the expression levels of AtLTPG1 transcripts are regulated by Abi, abiotic stress and stress hormones, 10-day seedlings were added to MS medium supplemented with 200 mM NaCl, 200 mM mannitol, 20% PEG, or 1 M ABA. Incubated, RT-PCR was performed. The gene actin7 (At5g09810) was used for qualitative and quantitative analysis of cDNAs. The rd29A gene (At5g52310), known as the drought stress inducible gene, was used as a control for the lack of water response. Expression of the rd29A gene increased significantly in response to water deprivation treatment, whereas expression of AtLTPG1 transcript was not affected by ABA, salt, or osmotic pressure (data not shown).

Of the AtLTPG1 gene Spatial and temporal expression is behind the promoter region of the AtLTPG1 gene Bacterial uidA genes were linked and introduced into Arabidopsis plants. Tissue samples of five independent transgenic plants were stained for investigation of GUS activity. Behind the promoter region of the AtLTPG1 gene, the GUS gene was strongly expressed on the ground of 10 day old seedlings. In addition, GUS expression was observed at the tip of the roots and at the top of the stylars, pollen, calyx and petals. Moreover, the long cell walls and seeds were stained in the early stages of development (before the seeds browned). When the stem and leaf tissues are cut into sections, the GUS gene is expressed in stems and leaf epidermis, including trichomes, leaf mesophyll cells, and stem cortex and xylem. Observation was made (FIG. 2).

Example 3 AtLTPG1 Protein Present in Plasma Membranes

GPI-anchor proteins are found primarily in the plasma membrane [Udenfriend S, Kodukula K (1995) Annu Rev Biochem 64: 563-591]. AtLTPG1 has a GPI-anchor region at the C-terminus to investigate the intracellular location of AtLTPG1. It was. To this end, vectors and targeting plasmids lacking targeting signals made by inserting the EYFP protein between the lipid transfer protein domain and the GPI-anchor domain (between 162 and 163 amino acid residues) were introduced into Arabidopsis protoplasts. Arabidopsis protoplasts transformed with AtLTPG1: EYFP vectors were observed under fluorescence microscopy, and fluorescence signals were observed on the plasma membrane (FIGS. 3C and D). However, fluorescent signals of Arabidopsis protoplasts transformed with the control EYFP vector were detected in the cytosol (FIGS. 3A and B).

The role of the GPI-anchor domain of AtLTPG1 protein in plasma membrane targeting was investigated. To this end, RFP was linked to the C-terminal end of the AtLTPG1 protein from which the 48 amino acids (145-193 residues) containing the GPI-anchor domain were removed from the C-terminus. When the AtLTPG1Δ48: RFP vector was co-introduced into protoplasts with Bip: GFP as an ER marker or ST: GFP vector as a Golgi marker [Min et al., (2007) Plant Physiol 143: 1601-1614], the red fluorescence signal was Bip: Although located together with the GFP signal (Figs. 3B-H), there was no ST: GFP signal (Fig. 3I-L). These results indicate that the AtLTPG1 protein is present in the plasma membrane and the AtLTPG1 protein is essential for GPI-encapsulation to leave the ER.

<Example 4. By T-DNA atltpg1  Isolation of Knock-Out Mutants>

In a plant to examine the functions of genes AtLTPG1, the atltpg1 (Garlic_1166_G01.b.1a.Lb3Fa) Arabidopsis mutant by the T-DNA was obtained from SAIL source (resources) (Fig. 4A). Seeds were germinated in 1/2 MS agar medium supplemented with phosphinothricin, and then PCR selection was confirmed by specifically amplifying the T-DNA insertion side DNA sequence in the AtLTPG1 coding region.

Garlic_1166_G01.b.1a.Lb3Fa plants were screened through insertion of T-DNA into the second exon of the AtLTPG1 gene by PCR using a left border (LB1) and AtLTPG-1 CDNA F2 primer. However, no PCR product was observed when DNA isolated from wild-type plants was amplified with this primer set. In addition, when PCR was performed using the AtLTPG-1 cDNA F2 and AtLTPG-1 cNDA R2 primers, the PCR product was consistent with the coding region and the intron of the AtLTPG1 gene was detected in the wild type, but in Garlic_1166_G01.b.1a.Lb3Fa plants It was not detected (FIG. 4B). Finally, when phosphinothricin-resistant Garlic_1166_G01.b.1a.Lb3Fa plants were back-crossed with the wild type to analyze phosphinothricin-resistance, the F ₂ progeny from the three independent plants were approximately 3 (resistance): 1 (sensitive) was separated (Table 2). These results indicate that the isolated Garlic_1166_G01.b.1a.Lb3Fa plant ( atltpg1 ) is a homozygous in which T-DNA is inserted into the second exon of the AtLTPG1 gene, and T-DNA is inserted into the AtLTPG1 gene in a single copy.

TABLE 2 Phosinothricin- resistance analysis of F ₂ progeny of atltpg-1 heterozygotes (F ₁ )

of the AtLTPG1 Gene in the atltpg1 Mutant RT-PCR was performed to isolate total RNA from 10 day old wild type and atltpg1 mutant seedlings to assess mRNA expression. As shown in FIG. 4C, wild-type RNA contained a full length AtLTPG1 transcript, whereas the atltpg1 mutant did not detect any AtLTPG1 transcript (FIG. C). Under normal growth conditions, the atltpg1 knockout mutant grew and developed normally compared to wild-type plants.

<Example 5. atltpg1  Reduction of cuticular wax load of mutants>

LTPs are associated with the transfer of wax or cutin monomers (Kader JC (1996) Annu Rev Plant Physiol Plant Mol Biol 47: 627-654), to assess whether disruption of the AtLTPG1 gene causes changes in the transport of extracellular lipids. Cuticle waxes extracted from stems, leaves and long shells of wild-type and atltpg1 mutants were analyzed by GC-Mass and GC. The total wax loading of stem, leaf and long shells of the atltpg1 mutant was reduced by 5 to 32% compared to wild type (Col-O). The largest decrease (30-50%) of C29 alkanes (nonacosine), the major component of stem and long cuticle wax, was observed in the mutants. In addition, C28 fatty acids and C24-C28 primary alcohols in stems, leaves and long shells of atltpg1 mutants were reduced by 12-50 % compared to wild type (FIG. 5). Although major changes in the cuticular hadeorago total carrying capacity of the wax is that the reduction in atltpg1 mutants, SEM (scanning electron microscopy) as observation cuticle of the stem and silique atltpg1 mutant external napjil crystal (epicuticular wax crystals) is observed (Not shown). The cuticle wax-deficient phenotype was restored when the atltpg1 mutant was supplemented with the introduction of an about 3.8 kb AtLTPG1 genomic clone. Indeed, the cuticle wax component comprising C29 alkanes and C24 to C28 primary alcohols was completely recovered in this plant (FIG. 5A).

Interestingly, the cutin monomers, the major component of extracellular lipids, in wild-type and atltpg1 mutants showed no significant difference in total amount and chemical composition (data not shown). Consistent with these results, atltpg1 leaves were used to determine the permeability of leaf cuticles, including polyesters of altered cuticles, to 0.05% toluidine blue-O, Bessire et al., (2007) EMBO J 26: 2158 -168] (data not shown).

<Example 6. atltpg1  Accumulation of intracellular lipid products and platoglobules in mutants>

The ultrafine structure of atltpg1 mutant stems and leaves was investigated by reducing wax loading in atltpg1 mutants. Briefly, wild type and atltpg1 mutant leaves and stems were fixed and embedded in LR white resin. Thin sections were stained and observed by transmission electron microscopy (TEM). Cytoplasmic protrusion into the vacuole was observed in the stem and leaf epidermal cells of the atltpg1 mutant (FIGS. 6A-D). In addition, the grana and stromal stratification of the stem cortex of the atltpg1 mutant and the chloroplasts of the leaf lobules cells were destroyed, and the size of the platoglobules was larger and more numerous (FIGS. 6E-J). Many uncertain portions stained with dark colors were detected in the cytosol and vacuoles of the epidermis and lobules of the atltpg1 mutant (FIGS. 6B, D and I). When enlarging the cuticle layer of the stem epidermis, wild-type cuticles were observed to be thin and continuous electron-dense, whereas mutant cuticles were destroyed and looked more scattered (FIGS. 6K and L).

Stem epidermal shells of wild-type and atltpg1 mutants were stained with Nile Red to identify darkly stained portions. The fluorescence signal was weakly detected in the cuticle layer of the wild-type epidermal shell, and autofluorescence was observed around the pores (FIGS. 7A-C). Conversely, high concentrations of lipids were observed in the mutant epidermal shells, and the integration of fluorescence signals and optical microscopy images showed that accumulation was consistent with the size and shape of the darkly stained portion, which has not yet been revealed (FIGS. 7D-). I).

Example 7 Fungal Pathogens Alternaria Brassicipola ( Alternaria brassicicola)  Increased sensitivity to atltpg1  Mutant>

Since changes in the cuticle layer structure were observed due to abnormal cuticle wax delivery in the atltpg1 mutant, we investigated how the response to the infection of Alternaria brassicicola , a necrotrophic fungus, was changed. . Col-0 has been used as a natural wild ecotype that is resistant to Alternaria brassica. When the leaves of the Arabidopsis larvae were infected by fungal spores, the spores began to germinate, forming appressoria and hyphal networks, penetrating into the host and eventually encroaching on tissues [McRoberts N, Lennard JH (1996). ) Plant Pathol 45: 742-752. As shown in FIG. 8A, Col-0 infected with Alternaria brassica cola formed a small brown gangrene injury site, but the atltpg1 plant formed a damaged site spread widely by infection. Lactophenol-aniline blue staining showed widespread damage sites severely eroded by fungal hyphae (FIG. 8B).

1 is an isolated AtLTPG1 gene. (A) Expression of AtLTPG1 transcript in stem (S) and stem epidermal cells (E). (B) AtLTPG1 protein amino acid sequence is shown. Asterisks indicate conserved cysteine residues. Signal peptide domains and GPI-anchor domains are underlined and double underlined, respectively. Arrows indicate the EYFP-insertion position for the intracellular position of AtLTPG1. (C) AtLTPG1 protein schematic. SP; Signal peptide domain, GPID; GPI-anchor domain.

2 shows GUS expression under AtLTPG1 promoter regulation in transformed Arabidopsis. (A) A 10 day old seedling. The arrow shows an enlarged photograph of the root end (A-1) and the start of the side root (A-2). (B) 2 week leaves. Arrows show enlarged photographs of the tricom (B-1). (C) leaf cross section, (D) stem, (E) cross section of stem, (F) flower, (G) carpel, (H) stamen, (I) petal, (J) calyx, (K) long wall and development Seeds being grown.

3 is a diagram showing that AtLTPG1: EYFP exists in the plasma membrane of Arabidopsis protoplasts. (A, B) control, p35S-EYFP-Flag / Strep plasmid, (C, D) AtLTPG1: EYFP vector, (E, I) AtLTPG1Δ48: RFP vector, (F) BiP: GFP vector, (J) ST: GFP vector. Images are obtained through GFP filters (A, C, F, J), RFP filters (E, I) or merging (G), (I) and (J) merging (E) and (F) ( K) was obtained. Images by light are B, D, H, I.

4 is atltpg1 tagged with T-DNA Mutant was isolated. (A) Genome constitution of the AtLTPG1 gene tagged with T-DNA. AtLTPG1 genomic DNA shows two exons (white box), one intron (black line) and UTRs (grey box). T-DNA direction is indicated by left (LB) and right (RB) borders. (B) PCR resulted from genomic DNA isolated from wild type (WT) and atltpg1 mutants. (C) RT-PCR analysis of the AtLTPG1 gene in wild type (WT) and atltpg1 mutants. Total RNA (0.2 mg) was used to detect AtLTPG1 transcript.

5 shows a cuticle wax loading composition of atltpg1 (gray) complemented with wild type (white), atltpg1 mutant (black) and AtLTPG1 genomic DNA. Cuticle waxes were extracted from stems (A), leaves (B), and long shells (C) of Arabidopsis plants, 5-6 weeks old. Each value represents six independent measurement standard deviations. Asterisks indicate statistical significance for wild type (P <0.05).

6 is a transmission electron micrograph of wild type and atltpg1 mutants. Wild type stem epidermal cells (A), stem epidermal cells of atltpg1 mutant (B). Wild type leaf epidermal cells (C) and atltpg1 mutants (D). Stem cortical chloroplasts of wild type (E) and stem cortical chloroplasts of atltpg1 mutant (F). (G) Closeup of (F). Leaf leaf vein chloroplast (H) of wild type and leaf leaf vein chloroplast (I) of atltpg1 mutant. (J) is a close-up photo of (I). Cuticle layer (K) of wild type and cuticle layer (L) of atltpg1 mutant. Bars are 2 mm, 2 mm, 2 mm, 1 mm, 0.5 mm, 0.2 mm, 0.2 mm, 1 mm, 1 mm, 0.5 mm, 0.2 mm, and 0.2 mm from (A) to (I), respectively. (Ch; chloroplast, cu; cuticle, cw; cell wall, m; mitochondria, pg; platogranules, sg; starch granules).

7 shows Nile red staining results of epidermal cells of Arabidopsis wild-type and atltpg1 mutants. (AC) wild type stem epidermis, (DF) atltpg1 mutant stem epidermis. The (GI) image is an enlargement of the white boxes of (D) to (F), respectively. Images were obtained using fluorescence microscopy (A, D, G), light microscopy (B, E, H), merging (A) and (B) and merging (C), (D) and (E) Figure (I) obtained by merging (F), (G) and (H).

Figure 8 shows disease development of atltpg1 mutants infected with Alternaria Brassicchia . (A) Necrotic damage site phenotype, (B) The photomicroscopic observation of the necrotic injury site after phenol-aniline blue staining.

9 is a diagram showing the degree of similarity (%) between the AtLTPG-1 gene and other plants. P. patens ; Lichen ( Physcomitrella patens subsp ), Picea sitchensis ; Sitka (wood), Vitis vinifera ; Grapes, Oryza sativa : rice, Arabidopsis thaliana ; Arabidopsis, Thlaspi caerulescens ; Alpine pennycres, Cicer arietinum ; Chickpea , Zea mays ; corn.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for regulating cuticular wax load amount in plant cell          wall and the plant <130> PN08239 <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 582 <212> DNA <213> Arabidopsis thaliana <400> 1 atgaagggtc ttcatctcca cctcgtccta gtcaccatga cgatcgttgc ctccatcgcc 60 gccgctgcac cggctgctcc cggaggagct ctggctgatg aatgcaacca ggattttcaa 120 aaggtgactt tgtgtttgga ctttgcgacc gggaaggcaa caattccgtc taagaagtgt 180 tgtgacgctg ttgaagatat caaagaaaga gatccaaagt gtttgtgttt cgttatacaa 240 caagcgaaga caggaggaca agccttgaag gatcttggtg ttcaagaaga caaactcatt 300 caactcccaa cttcttgtca gctccacaac gctagcatta ccaactgtcc aaagcttcta 360 gggatttcac ctagctcgcc agacgcagcc gtattcacaa acaatgccac aacaacaccg 420 gtggcaccag caggaaagtc tccggcaact ccagctacgt ccacggataa gggaggatca 480 gcttcagcaa aggatggtca cgcagtcgtt gctctagccg tcgctttgat ggccgtctcc 540 tttgtcttga ccttgcctag acatgtcaca ttagggatgt aa 582 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gccttttcag aaatggataa atagccttgc ttcc 34 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gcccgggata atgaagggtc ttcat 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cggatcctta catccctaat gtgac 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 catccaagct gttctctcct tgtac 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cagacactgt acttcctttc aggtg 25 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cgcggttctt gagaagaccg gtgtga 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gagcttctcc agctatcgcc attagg 26 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cggatcctga taatgaaggg tcttca 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cggatccaca tccctaatgt gacatg 26 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctctagaagg atacgtagac aattggtag 29 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gcccggggct tgttgaagat cttgtttg 28 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 acccggggga cttgggattg gctgaggtt 29 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggggcaggat ctcctgtcat ctc 23 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcgctgcgaa tcgggagcgg cg 22 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggccggcctg gaggtggagg tggagctgtg agcaagggc 39 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggccccagcg gccgcagcag caccagcagg gtccttgtac agctc 45 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tccacctcca cctccaggcc ggcctgaagc tgatcctccc tt 42 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggtgctgctg cggccgctgg ggccgcaaag gatggtcacg ca 42  

Claims (13)

T-DNA was inserted into Arabidopsis thaliana Lipid Transfer Proteins GPI-anchored (AtLTPG1) gene, which consists of the nucleotide sequence of SEQ ID NO: 1, thereby reducing C29 alkanes by 30-50% compared to Arabidopsis wild type, and C28 fatty acid. And dysfunctional mutant Arabidopsis plants with a 12-50% reduction in C24-C28 primary alcohol. delete delete delete By inserting T-DNA into the AtLTPG1 gene derived from Arabidopsis nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 1, C29 alkanes are reduced by 30-50% compared to Arabidopsis wild-type, and C28 fatty acids and C24-C28 primary alcohols are reduced. A method of controlling the loading of cuticle wax in a cell wall comprising the step of preparing a 12-50% reduced malfunction mutant Arabidopsis plant. delete delete delete The method of claim 5, wherein the plant has an increased sensitivity to Alternaria brassicicola. delete delete delete delete

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