KR20150125399A - Peroxidase genes controlling plant growth, development and lignin content, Vector containing a peroxidase RNAi cassette, and Transformant containing the vector thereof - Google Patents

Abstract

The present invention relates to peroxidase genes controlling growth, development, and lignin content by light and brassinosteroid, to a vector including a peroxidase RNAi cassette, and to a transformant containing the vector.

Description

A vector comprising a peroxidase gene, a peroxidase RNA interference cassette for regulating plant growth and lignin content, and a transformant into which said vector has been introduced (a plant containing peroxidase genes controlling development and lignin content, a vector containing a peroxidase RNAi cassette, and Transformant containing the vector thereof.

The present invention relates to a vector comprising a peroxidase gene, a peroxidase RNAi cassette, which regulates plant growth development and lignin content by light and brassinosteroids, and a transformant into which said vector is introduced.

Plants are not only aware of light using photoreceptors that recognize light, but are also controlled through interactions with a variety of environmental factors, including growth hormone. Studies on plant hormones and light have been extensively studied in order to improve crop productivity. Brassinosteroid, a plant growth-promoting steroid hormone, regulates many aspects such as plant growth and development. In addition, it has various physiological control functions such as cell growth, division, and cell wall differentiation. Recently, it has been proved that photoreactivity of plant is closely related to brassinosteroid. However, molecular mechanisms for the interaction of light and brassinosteroids, and the functioning of signal transduction mediators and target genes for various light conditions, are still lacking.

Plants have the ability to cope with and adapt to various environmental changes by regulating growth and development. Through the entire plant life cycle from seed germination to flowering, the plant development program is known to increase the survival suitability of plants through constant interaction with environmental stimuli. The regulation of the development of plants by such environmental stimuli is mainly caused by the interaction with the external environment and the intrinsic developmental programs of plants, and it is known that plant hormones mainly perform functions essential for such interactions.

Peroxidases are multifamilies composed of various isozymes and isoforms and are classified into Class I, II, and III according to their reaction specificity and structure (Welinder, 1992). Class I peroxidase is an intracellular enzyme, Class II peroxidase is an extracellular enzyme including lignin and manganese-dependent peroxidase. Class III peroxidase (EC 1.11.1.7; Prxs) is a plant-specific and exogenous secreted or vacuolated enzyme that affects many physiological processes with the whole plant life cycle due to many isomers and various expression control mechanisms . Peroxidative and hydroxylic catalytic reactivity enables synthesis of reactive oxygen species and cell wall components. It is also associated with aging, including plant development, differentiation, growth, lignification, suberization, auxin degradation and oxidation of indoleacetic acid, cell wall protein cross-linking, defense against pathogen infiltration, It is responsible for various functions (Hiraga et al., 2001). Class III peroxidase plays a diverse role in the life cycle of plants due to the presence of multiple isoforms in plants and low substrate specificity, but their overall signaling mechanisms have not been elucidated. Because of these properties, the function of the 73 species of periosteal peroxidase is not fully understood.

The inventors of the present invention have been studying the molecular mechanism of the interaction between light and brassinosteroids. Among the Arabidopsis peroxidase genes, Prx2, Prx8, Prx35, and Prx73 genes are involved in growth and lignin by light and brassinosteroids And the present invention was completed.

Accordingly, it is an object of the present invention to identify the nucleotide sequence and function of a peroxidase gene that regulates plant growth and lignin content by light and brassinosteroids.

In order to accomplish the above object, the present invention provides a peroxidase gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

The present invention also provides a vector comprising an RNA interference (RNAi) cassette for the peroxidase gene.

The present invention also provides a transformant into which the vector is introduced.

Since the peroxidase gene of the present invention has a function of regulating plant growth by light and brassinosteroids, it is possible to provide a crop having increased productivity and functionality by controlling the expression of these genes by manipulating these genes.

Specifically, the transgenic plants knocked down the expression of these genes not only elongate their hypocotyls and roots but also grow rapidly in leaf size, length of petiole, and bolting time, . Therefore, the use of this technology in economic crops such as flower crops can promote the growth and flowering and speed up the seed production period, so that it is possible to provide the crops with excellent productivity and commerciality.

In addition, the transgenic plants knocked down the expression of these genes are inadequate to support the wild type, because the components of the cell wall, especially the lignin content, are reduced and the overall mechanical supportability is lowered. The use of this technology in the development of bioenergy crops has the advantage of reducing the lignin content and softening the cell walls of the plant, thereby reducing the chemical treatment costs of the woody part in the pulping and bio-energy extraction stages.

In addition, the transgenic plants knocked down the expression of these genes can be confirmed to have reduced disease resistance. Conversely, overexpression of this gene in a specific tissue such as a leaf can increase the physical resistance of the cell and increase the disease resistance.

Figure 1 shows the selection of peroxidase genes (Figure IA) co-regulated with brassinosteroids and light through microarrays and phylogenetic trees (Figure 1B) with 73 species of Arabidopsis thaliana peroxidase amino acid sequences.
2A is a diagram of a transformation vector (RNAi) used to inhibit the expression of a peroxidase gene in a plant. Figure 2B is a simplified schematic diagram of each peroxidase recombinant vector 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi, 35Spro: Prx73 RNAi.
FIG. 3 is a diagram showing the number of replications of chi - square transfected genes of 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi and 35Spro: Prx73 RNAi transformants.
Fig. 4 shows the homozygous line of 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi, and 35Spro: Prx73 RNAi transformants, which were 6 days old, (FIG. 4A) and their relative length analysis (FIG. 4B), and the level of transcriptional repression inhibition (FIG. 4C) of each peroxidase gene through qRT-PCR. The scale bar in the image = 1 mm.
FIG. 5 is a graph showing peroxidase activities of 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi and 35Spro: Prx73 RNAi transformant homozygous line for 6 days.
FIG. 6 is a graph showing the H ₂ O ₂ Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi, and 35Spro: Prx73 RNAi were inoculated in a medium containing 0.1% 3,3'-diaminobenzidine (DAP) (A). Fig. B is the upper layer, C is the cotyledon, and D is the diagram showing the accumulation of hydrogen peroxide as a result of staining the root portion with DAP.
FIG. 7 is a view showing the bolting time in the 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi, and 35Spro: Prx73 RNAi transformants grown in the long-day conditions.
Fig. 8 is a picture showing the phenotype of 35Spro: Prx2 RNAi transformant at 30 days. A and B are phenotypes of wild-type and 35Spro: Prx2 RNAi transformants. C is the 5th, 6th, 7th and 8th leaves except the cotyledons, D, E and F are the petiole, Lt; / RTI > In the image, the scale bar = 2 cm.
Fig. 9 is a picture showing the phenotype of 35Spro: Prx8 RNAi transformant 30 days old. A and B are phenotypes of the wild type and 35Spro: Prx8 RNAi transformants. C is the 5th, 6th, 7th and 8th leaves except the cotyledons, D, E and F are the petiole, FIG. In the image, the scale bar = 2 cm.
FIG. 10 is an analysis of the phenotype of 35Spro: Prx35 RNAi transformant 30 days old. A and B are phenotypes of the wild type and 35Spro: Prx35 RNAi transformants. C is the 5th, 6th, 7th and 8th leaves except the cotyledons, D, E and F are the petiole, FIG. In the image, the scale bar = 2 cm.
Figure 11 is a plot showing the erosion and size (Figure 7A) and petal comparisons of the 6-week wild-type and 35Spro: Prx35 RNAi transformants. The scale bar in the image = 1 cm.
Fig. 12 is an analysis of the phenotype of the 4-week-old 35Spro: Prx73 RNAi transformant. A and B are phenotypes of wild-type and 35Spro: Prx73 RNAi transformants. C is the 5th, 6th, 7th and 8th leaves except the cotyledons, D, E and F are the petiole, FIG. In the image, the scale bar = 2 cm.
Figure 13 is a graph showing the relative chlorophyll content of the 6.5 wild-type and 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi, and 35Spro: Prx73 RNAi transformants.
Fig. 14 is a graph comparing the degree of accumulation of lignin in a phytohormone-HCl stained oocyte with the cross section of the stem of the wild-type and 35Spro: Prxs RNAi transformants stained with Toluidine blue. A. Arabidopsis wild type B. 35Spro: Prx2 RNAi C. 35Spro: Prx8 RNAi D. 35Spro: Prx35 RNAi E. 35Spro: Prx73 RNAi A, B, C, D, E left: toluidine blue staining A, B, C, D , E right: phloroglucinol-HCl staining

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

<Examples>

A. Materials and Experimental Methods

One. Microarray  Data acquisition and analysis methods

Microarray experiments were performed using the Affymetrix ATH1 GeneChip system. The plants of the wild type Ws-2, bri1-5, phyB-77 and bri1-5 phyB-77 were grown in 0.5 MS plate (0.5 Murashige-Skoog salts, 1% sucrose; pH 5.75) for 7 days under the red light, Sampling was performed. As a result of the microarray experiment, 12 different CEL files were created and all results were submitted to the Gene Expression Omnibus (GEO) ( http://www.ncbi.nlm.nih.gov/projects/geo/ ) under accession number GSE46456 Statistical analysis of the results was performed according to the method described previously (Chung et al., 2011).

2. Plant material and growing condition

Arabidopsis seeds were seeded on surface-sterilized MS plates and treated for 3 days at 4C before growing the seedlings in a 22C environment under long-day conditions (16 hours nominal / 8 hours memorization) and continuous red or white light conditions.

In order to introduce the 35Spro: PRX RNAi construct into the wild type, the coding sequence of each gene was cloned into pENTR / SD / D_TOPO (Life Technologies, http://www.lifetechnologies.com ) to analyze the base sequence, A vector was constructed to allow effective gene silencing by forming hairpin RNA using the vector pB7GWIWg2 (II) vector.

Recently, a method to induce gene silencing by RNA interference (RNAi) has been developed and studies have been actively conducted on the construction of dsRNA expression vector, RNAi and target alignment, efficiency of RNAi and detection of RNAi . RNAi is a phenomenon in which double-stranded RNA (dsRNA) is introduced into an organism or cell and degrades homologous mRNA. Recently, RNAi has been widely used as a tool to knock out the expression of specific genes in various organisms.

Transgenic plants transformed with knockout of peroxidase factors were prepared using Arabidopsis transformants with the vectors introduced therein and the RNAi cassettes. Finally, the resulting plasmid was introduced into Agrobacterium tumefaciens (GV3101) to induce Arabidopsis transformation by the floral dip method (Clough and Bent, 1998). The transformed seeds were screened for independent transgenic plants in 0.5X MS medium containing Vasta.

3. Plasmid preparation

3-1. 35 Spro : PRX2 RNAi  Vector manufacture

Approximately 150 ng of Arabidopsis Col-0 cDNA was obtained as a PCR product using the AtPrx2 forward primer (5'-CACCATGGCGATCAAGAACATTCTCGC-3 ') and the reverse primer (5'-GTTAGGGAAGGC GCATCTCTTC-3') as template.

PCR was performed for 30 cycles (94 30 sec; 59, 30 sec; 72, 1 min) using 1 unit of nPfu-Taq (Enzynomics). The obtained PCR products were obtained by removing slices from 1% agarose, II gel extraction kit (QIAGEN).

The PCR fragments obtained for use in the Gateway system were first subjected to recombination reaction in an entry vector pENTR / SD / D_TOPO (Life Technologies, http://www.lifetechnologies.com ) to prepare plasmids and E. coli ') And grown on LB plates containing 100 μg / μl of kanamycin to obtain a single colony.

The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmid and confirmed the insertion as a restriction enzyme.

Through sequencing, it was confirmed that the identified plasmid contained the 982 bp AtPrx2 gene including the ORF (open reading frame).

The isolated plasmid was finally inserted into a pB7GWIWG2 (II) RNAi vector (invitrogen) corresponding to the destination vector to prepare a plasmid.

As described above, 35Spro: Prx2 RNAi was prepared using the cloned AtPrx2.

3-2. 35 Spro : PRX8 RNAi  Vector manufacture

Approximately 150 ng of Arabidopsis Col-0 cDNA was obtained as a PCR product using the AtPrx8 forward primer (5'-CACCATG AGGGCAATCGCAGCTTGG-3 ') and the reverse primer (5'-GTTGTTGAAGGCTCTGCAGTTTGT-3') as template.

PCR was carried out with 1 unit of nPfu-Taq (Enzynomics) for 30 cycles (94 30 sec; 60, 30 sec; 72, 1 min) II gel extraction kit (QIAGEN).

The PCR fragments obtained for the Gateway system were first subjected to recombination reaction in an entry vector pENTR / SD / D_TOPO (Life Technologies, http://www.lifetechnologies.com), plasmids were prepared, and E. coli (Top 10 ') And grown on LB plates containing 100 μg / μl of kanamycin to obtain a single colony.

The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmid, and it was confirmed whether the plasmid was inserted as a restriction enzyme.

Through sequencing, it was confirmed that the identified plasmid contained the 982 bp AtPRX8 gene including the ORF (open reading frame).

Finally, a plasmid was prepared by inserting it into a pB7GWIWG2 (II) RNAi vector (invitrogen) corresponding to a destination vector.

As described above, 35Spro: Prx8 RNAi was prepared using the cloned AtPrx8.

3-3. 35 Spro : PRX35 RNAi  Vector manufacture

Approximately 150 ng of Arabidopsis Col-0 cDNA was obtained as a PCR product using the AtPrx35 forward primer (5'-CACCATG GCTCGCTTCGATATTGTTC-3 ') and the reverse primer (5'-GTTAAACGCACCACAATCACGACG-3') as template.

PCR was carried out using 1 unit of nPfu-Taq (Enzynomics) for 30 cycles (94 30 sec; 58, 30 sec; 72, 1 min). The obtained PCR products were obtained by removing slices from 1% agarose, II gel extraction kit (QIAGEN).

The PCR fragments obtained for use in the Gateway system were first subjected to recombination reaction in an entry vector pENTR / SD / D_TOPO (Life Technologies, http://www.lifetechnologies.com ) to prepare plasmids and E. coli ') And grown on LB plates containing 100 μg / μl of kanamycin to obtain a single colony.

The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmid and confirmed the insertion as a restriction enzyme.

Through sequencing, it was confirmed that the identified plasmid contained the 991 bp AtPRX35 gene containing the ORF (open reading frame).

Finally, a plasmid was prepared by inserting it into a pB7GWIWG2 (II) RNAi vector (invitrogen) corresponding to a destination vector.

35Spro: Prx35 RNAi was prepared using the cloned AtPrx35 as described above.

3-4. 35 Spro : PRX73 RNAi  Vector manufacture

Approximately 150 ng of Arabidopsis Col-0 cDNA was obtained as a PCR product using the AtPrx73 forward primer (5'-CACCATG GCGCGGTTCAGTCTGGTT-3 ') and the reverse primer (5'-GTTAAAGGCACCACAGTCACGAC-3') as template.

PCR was carried out with 1 unit of nPfu-Taq (Enzynomics) for 30 cycles (94 30 sec; 60, 30 sec; 72, 1 min) II gel extraction kit (QIAGEN).

The PCR fragments obtained for use in the Gateway system were first subjected to recombination reaction in an entry vector pENTR / SD / D_TOPO (Life Technologies, http://www.lifetechnologies.com ) to prepare plasmids and E. coli ') And grown on LB plates containing 100 μg / μl of kanamycin to obtain a single colony.

The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmid and confirmed the insertion as a restriction enzyme.

Through sequencing, it was confirmed that the identified plasmid contained the 991 bp AtPrx73 gene including the ORF (open reading frame).

Finally, a plasmid was prepared by inserting it into a pB7GWIWG2 (II) RNAi vector (invitrogen) corresponding to a destination vector.

As described above, 35Spro: Prx73 RNAi was prepared using the cloned AtPrx73.

4. Analysis of gene expression of each transformant

A six-day old plant of a homozygote of 35Spro: Prx2 RNAi (pH7GWIWGII: Prx02), 35Spro: Prx8 RNAi (pB7GWIWGII: Prx08), 35Spro: Prx35 RNAi (pB7GWIWGII: Prx35), 35Spro: Prx73 RNAi (Fermentas, now Thermo Scientific, http://www.thermoscientificbio.com/fermentas ), and the same amount of reverse transcribed material was subjected to real-time quantitative PCR (PCR).

qRT-PCR was performed by using each transformant cDNA as a template and using each gene specific primer (Prx2 qPCR forward: 5'-CGAAATTGAACGATGCATTGCTAAA-3 'and Prx2 qPCR reverse direction: 5'-CCA TGTTCAGTGAGGTTCTGAAAT- -GGATCCTAA AATGGACAGCAAACTGA-3 'and Prx8 qPCR reverse direction: 5'-AAATCTGACACAATCGACCTGGTTGAT-3'; Prx35 qPCR forward: 5'-CGAAGGGAACTTACCAGGACCTT-3 'and Prx35 qPCR reverse direction: 5'-TTGTCAAACGTCTTGGGCGTGAC- (KapaBiosystems, http://www.kapabiosystems.com ) together with the CCGGACCAAATAACAAAGTTACAGAA-3 'and Prx73 qPCR reverse direction: 5'-GCGGTTACGAAAGCCTTGTTGAAA-3') in a final volume of 20 l.

Samples were quantitated using standard curves of the results obtained with serially diluted control DNA, and each cDNA was normalized using UBQ10 (AT4G05320).

5. Peroxidase  Active measurement

Peroxidase activity was measured according to Chance and Maehly (1955) method (2001) using guaiacol (sigma) as substrate. 50 mM sodium acetate buffer (pH 7), 25 mM guaiacol and 25 mM H 2 O 2 were prepared and absorbance changes were measured at 470 nm.

6. DAB  (3,3'- diaminobenzidine ) dyeing

To analyze the accumulation of hydrogen peroxide (H ₂ O ₂ ), staining was performed with 0.1% 3,3'-diaminobenzidine (DAB; Sigma) using thordal Christensen (1997) . After DAB staining, the leaves were boiled in 95% ethanol for 10 minutes and washed twice with 50% ethanol to remove chlorophyll completely. The sample was fixed in 50% glycerol and photographed under an optical microscope.

7. Growth and Development Survey

PrS35: Prox8 RNAi (pB7GWIWGII: Prx08), 35Spro: Prx35 RNAi (pB7GWIWGII: Prx35), 35Spro: Prx73 RNAi (pB7GWIWGII: Prx02) Prx73) homozygous line and Arabidopsis wild-type (Col-0) seeds were seeded in 0.5XMS medium and cultivated for 6 days in a 22C long day condition (16 hour nominal / 8 hour memorization) Hypocotyls and root lengths were measured using software (Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, Texas). The length of petiole, leaf width and leaf length of the 5th leaf, including cotyledon, were measured by using the UTHSCSA image tool software for 4 weeks of wild type and transgenic plants, respectively. Lt; / RTI &gt; In addition, the length of the trunk was measured using image tool software for the 6 - week - old wild type and transgenic plants.

8. Chlorophyll content measurement

Chlorophyll contents were measured by using Lichtenthaler (1987) method. The chlorophyll contents were measured at 663 nm and 648 nm after 6 weeks of extraction.

9. Histochemical staining

Stems were fixed in FAA solution (10% v / v formaldehyde, 5% v / v acetic acid and 50% v / v ethanol) , 100%, each hour). The blocks were then transferred to a Peel-A-Way disposable embedding mold (Polysciences). Technovit 7100 and paraffin were cut into microtome (Microm). Lignin staining was stained by Chapple (1992) method and stained with toluidine blue.

B. Experimental Results

1. Arabian Pole Peroxidase gene  Selection

Microarray gene expression analysis of gul2 (phyB) mutant and brassinosteroid receptor mutant mutants deficient in photoreceptor phytochrome B showing hypodense extension under treatment with brassinosteroid biosynthesis inhibitor was diagnosed. As a result, the expression of peroxidase (Prx) genes was relatively decreased in the gul2 (phyB) mutant (FIG. 1A). Using the amino acid sequences of the 73 Arabidopsis genes in the NCBI GenBank and Phytozome ( www.phytozome.net ), we constructed a phylogenetic tree to analyze the peroxidase gene homologous to the selected peroxidase And that homology between Prx35 and PRX73 was high (Fig. 1B).

2. The Arabian Pole Of the peroxidase gene RNAi  Preparation of expression-inhibiting vector and preparation of said vector Using the transformed plant

The peroxidase recombinant vector, 35Spro: Prx2 RNAi (pH7GWIWGII: Prx02), 35Spro: the peroxidase recombinant vector, was downregulated in the photoreceptor mutation (phyB) but upregulated in the brassinosteroid defective mutation (bri1-5) Prx8 RNAi (pB7GWIWGII: Prx08), 35Spro: Prx35 RNAi (pB7GWIWGII: Prx35), and 35Spro: Prx73 RNAi (pB7GWIWGII: Prx73) (FIG. The constructed construct was introduced into Agrobacterium (GV3101) and transformed into Arabidopsis wild type. In the mRNA transcription after transfection, the target gene inserts each peroxidase gene fragment in the direction of sense and antisense to form the hairpin RNAi structure, and the endogenous intron (intron) The use of pB7GWIWGII and pH7GWIWGII vectors to be located can be usefully applied to highly efficient gene silencing using RNAi (Fig. 2A). pB7GWIWGII and pH7GWIWGII are microorganism selection markers, spectinomycin, and plant transformation markers, respectively, with Basta and hygromycin resistance genes.

In T2 generation, a transformant containing 1 copy of T-DNA was obtained by chi-square test (FIG. 3). These lines were first screened for surviving lines of all the transgenic markers of each gene vector in the T3 generation. Total RNA was isolated from each plant, and gene silencing using RNAi was used to screen for expression of the peroxidase (Prx02, Prx08, Prx035, Prx073) genes introduced into the transgenic Arabidopsis lines. Real-time PCR was performed using primers as primers, and pure lines of 35Spro: Prxs RNAi traits were selected.

An increased rate of growth and development in the oily plant stage was shown for 20 days of 6 days old plants of selected T4 generation 35Spro: Prxs RNAi transformants (Fig. 4A). The ratio of 35Spro: Prx2 RNAi to that of wild type was 78.7% and 33.2%, 35Spro: Prx8 RNAi was 31.8% and 22% respectively, 35Spro: Prx35 RNAi was 61% and 70.1% respectively, 35Spro: Prx73 RNAi was increased by 70.8% and 26.9%, respectively (Fig. 4B). Root length was longer than wild type, and hypocotyl growth developed rapidly. As a result of performing qRT-PCR by sampling them, it was confirmed that the transcriptional expression of each gene was inhibited as compared with the wild type (FIG. 4C).

3. Arabic Pole Peroxidase of peroxidase transformants  Active measurement

The peroxidase activity of each 35Spro: Prxs RNAi strain on 6 days was measured more than 3 times, and it was confirmed that all lines were reduced compared to the wild type (FIG. 5).

DAB (3,3'-diaminobenzidine) staining was performed on two-week-old wild-type and transformants in order to visualize the degree of hydrogen peroxide accumulation in transformants inhibited by transcription expression of each peroxidase gene. DAB is rapidly absorbed and polymerized by plants when it is H ₂ O ₂ and peroxidase, and it is stained in brown. Even in the normal state, 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi and 35Spro: Prx73 Accumulation was increased in RNAi transformants (Fig. 6A). Especially, cotyledons of transgenic plants were analyzed for brown spots, which are the reaction characteristics of DAB and H ₂ O ₂ , and accumulated high in leaf vein veins and vascular bundles. The pattern of cotyledon vein of the transformant is different from that of the wild type and the development of vein is decreased in 35Spro: Prx2 RNAi, 35Spro: Prx8 RNAi, 35Spro: Prx35 RNAi transformant cotyledon (Fig. 6B, C , D). Since the peroxidase catalyzes the reaction of oxidizing various substrates using hydrogen peroxide, the transformant promotes growth rather than the wild type, and the expression of peroxidase is inhibited in the RNAi transformant. Therefore, H ₂ O ₂ seems to accumulate more hydrogen peroxide than wild type.

4.35 Spro : Prx2RNAi Plant

The 35Spro: Prxs RNAi transformants are faster than the wild type and have fast stem growth (Fig. 7).

The initial growth rate was also increased, but the 35Spro: Prx2 RNAi transformant at 30 days was analyzed. As a result, the size of the 35Spro: Prx2 RNAi transgenic plant was increased compared to the wild type, and not only the leaf size, It is confirmed that the length is increased (A, B, C in Fig. 8). In order to observe the differences between the wild type and 35Spro: Prx2 RNAi transgenic plants, the plots, leaf length and leaf width length results of the fifth leaf, excluding the cotyledons, were plotted in more than 52 plants, , And 1.2 times, respectively (D, E, F in Fig. 8). The 35Spro: Prx2 RNAi transformants showed overall rapid growth.

5.35 Spro : Prx8RNAi Plant

Analysis of 35Spro: Prx8 RNAi transformants at 30 days showed that the size of 35Spro: Prx8 RNAi transgenic plants was increased compared with the wild type, and that not only the size was about twice as high but also the petiole, leaf area and leaf width of rosewater were increased (A, B and C in Fig. 9). In order to observe the difference between the wild type and 35Spro: Prx8 RNAi transgenic plants, the graphs of the petiole, leaf length and leaf width of the fifth leaf, except the cotyledon, were plotted in more than 52 plants, 1.6 times and 1.2 times , And 1.3 times, respectively (D, E, F in Fig. 9). 35Spro: Prx8 RNAi transformants were found to exhibit rapid growth overall.

6.35 Spro : Prx35RNAi Plant

Analysis of the 35Spro: Prx35 RNAi transformant at 30 days showed that the 35Spro: Prx35 RNAi transgenic plant size was increased compared to the wild-type strain, and that the overall leaf length was similar but the leaf length was increased (A, B and C in Fig. 10). Interestingly, however, 35Spro: Prx35 RNAi transgenic plants all showed a tendency to lie down. As a result of cutting the stem longitudinal section, it was confirmed that stem stem diameter was smaller than that of wild type (left side A and D in Fig. In order to observe the differences of wild type and 35Spro: Prx35 RNAi transgenic plants in more detail, the length of petiole, leaf length and leaf width of the fifth leaf, except cotyledon, were measured in more than 52 plants. And the length of the leaf width was increased or decreased by 1.3 times, respectively (D, E, F in Fig. 10).

The 35Spro: Prx35 RNAi transformants exhibited rapid growth overall. Analysis of the silage size of the 6-week wild-type and 35Spro: Prx35 RNAi transgenic plants revealed that the transformants were 1.4 times smaller, (Fig. 11).

7.35 Spro : Prx73RNAi Plant

The initial growth rate was also increased, but the 35Spro: Prx73 RNAi transformants were analyzed at 30 days. As a result, the size of 35Spro: Prx73 RNAi transgenic plants was increased compared with the wild type, and not only the leaf size, (Fig. 12A, B, C). In order to observe the difference between the wild type and 35Spro: Prx73RNAi transgenic plants in more detail, the petiole, leaf length and leaf width length of the fifth leaf, excluding cotyledon, were measured and plotted in a graph. As a result, 1.6 times and 1.3 times , And 1.3 times, respectively (D, E, F in Fig. 12). The 35Spro: Prx73 RNAi transformants exhibited rapid growth overall, and the timing of the emergence of the phloem was also faster than that of the wild type.

These results suggest that peroxidase 2, 8, 35, and 73 are involved in the development of Arabidopsis thaliana.

8.35 Spro : PrxsRNAi Chlorophyll content of plants

Aging was accelerated in the 6S35Spro: Prxs RNAi transformants compared to the wild type, and leaf sulphation was generally observed in the leaves, but 35Spro: Prx35 RNAi # 12-6 was weakened. The chlorophyll content of 3 repetitions was 30-50% lower than that of the wild type except for 35Spro: Prx35 RNAi # 12-6 line, and peroxidase 2, 8, and 73 were found to be involved in the regulation of senescence ).

9. 35 Spro : PrxsRNAi Lignin analysis of plant

Peroxidase is involved in ligninization and reactive oxygen species metabolism, and it is known that most of these genes are expressed in 6-week-old peroxidase plants. It is also reported to be expressed in developmental stages and stress conditions. Lignification of plant cell walls is increased in response to environmental stresses, which is associated with reduced plant growth and structural integrity of the tissue. Lignin is a good specimen for the development of dry tolerant plants by controlling the ligninization by relating to the dry tolerance, including reducing the intracellular transpiration during dry stress, including physical defense against pathogen infection. It is also associated with various abiotic stress defense mechanisms such as low temperature and photoreaction (Denness et al., 2011).

Transgenic plants were stained with toluidine blue and phloroglucinol-HCl, a lignin-staining reagent, to observe the cross-section of the stem. The patterns of the vascular tissues were different from those of the wild type. In the case of the wild type, the number of stem vascular bundles is generally known to be 6-8, but the 35Spro: Prx35 RNAi line is observed in five, the pith specific gravity is decreased and the arrangement is irregular. In addition, they showed phenotypes in which the plant tissues were slowed down, inhibited the production of interfascicular fibers, weakened the supportive function of the plants, and layed up the plants. The 35Spro: Prx8 RNAi line also has five vascular bundles and the 35Spro: Prx73 RNAi line has slowed the development of bulb tissue (Fig. 14). The lignin content is also reduced overall compared to the wild type. The anatomical observation of the cross-section of BR-associated mutant stalk of Arabidopsis shows that mutant has slowed the development of vaginal tissue while the development of vaginal tissue is relatively unaffected. Based on these phenotypes, it is likely that these peroxidase genes are involved in the ongoing research on the differentiation and formation of bulbocytes through the relationship with lignin and brassinosteroids as new genes involved in lignin synthesis .

10. Statistical significance

In all charts, asterisks show statistically significant differences by t-test with the following probabilities compared to the control: * p <0.005

<References>

Chance B and Maehly AC (1955) Assay of catalases and peroxidases. Methods Enzymol 2: 764775.

Chapple CC, Vogt T, Ellis BE and Somerville CR (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell 4: 1413-1424.

Auxin-stimulated DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis. In this study, we investigated the expression of Auxin-stimulated DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis thaliana. Plant J 66: 564578.

Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735743.

Denison L, McKenna, JF, Segonzac C, Wormite, Madhou P, Bennett M, Mansfield J, Zipfel C and Hamann T (2011) Cell wall damage-induced lignin biosynthesis is regulated by a ROS and jasmonic acid dependent process in Arabidopsis thaliana . Plant Physiol. 156: 13641374.

Hiraga S, Sasaki K, Ito H, Ohashi Y and Matsui H (2001) A large family of class III plant peroxidases. Plant Cell Physiol 42: 462-468.

Lichtenthaler HK (1987) Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol 148: 350-382.

Neff MM and Chory J (1998) Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during Arabidopsis development. Plant Physiol 118: 27-36.

Thordal-Christensen H, Zhang Z, Wei Y and Collinge DB (1997) Subcellular localization of H ₂ O ₂ in plants. H ₂ O ₂ accumulation in papillae and hypersensitive response during the barleypowdery mildew interaction. Plant J 11 (6): 1187-1194.

Welinder KG (1992) Superfamily of plant, fungal and bacterial peroxidases. Curr Opin Struct Biol. 2: 388-393.

<110> SNU R & DB FOUNDATION <120> Peroxidase genes controlling plant growth, development and lignin          , a vector containing a peroxidase RNAi cassette, and          Transformant containing the vector thereof <130> snu-20140430-prx <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 978 <212> DNA <213> Arabidopsis <400> 1 atggcgatca agaacattct cgcccttgtg gttcttctta gcgtggttgg agtttctgtc 60 gccattccac agttgcttga cctcgactac taccggtcta agtgtcccaa ggcagaggaa 120 attgttcgtg gtgtcacagt acaatatgtt tctcgccaga aaacccttgc cgctaaactt 180 ctaaggatgc atttccatga ttgtttcgtc agaggatgtg atggttccgt tcttctgaaa 240 tctgcaaaga atgatgcgga aagagacgct gtccccaacc tgaccctgaa aggttatgaa 300 gtggtggatg cggccaagac agcgctggag aggaagtgtc ctaatctcat ttcttgcgct 360 gatgttcttg ccttggtcgc cagagatgcc gtggcagtga tcgggggacc atggtggccg 420 gttccattgg gccgcaggga tggacgcatc tcgaaattga acgatgcatt gctaaattta 480 ccatctcctt tcgccgacat aaagacgctg aagaagaact ttgccaacaa gggtcttaac 540 gctaaagacc ttgtggttct ctcagggggt cacaccattg gaatctctag ttgcgctctc 600 gtcaacagtc gtctctacaa cttcacagga aagggcgatt ctgacccatc catgaaccct 660 agctacgtga gggaattgaa gagaaagtgc ccgcctacag atttcagaac ctcactgaac 720 atggacccag gcagtgcgtt gacattcgac actcactact tcaaggtcgt ggctcagaag 780 aaagggctct tcacatctga ctctacgctt ctcgatgaca ttgagaccaa aaactacgtt 840 cagactcagg ccattctccc tcctgtgttt tcttctttca ataaagattt ctccgattcc 900 atggtcaaac ttggtttcgt ccaaattctt accggcaaaa atggtgagat caggaagaga 960 tgcgccttcc ctaactaa 978 <210> 2 <211> 933 <212> DNA <213> Arabidopsis <400> 2 atgagggcaa tcgcagcttg gtttttcatt ttctgttacc ttgttccctc tgtttttgcg 60 cagctccggc atgggtttta cgagggtaca tgccctcccg ctgaatctat cgtgggtaga 120 gttgttttca accattggga ccgtaaccga acggttactg cagcattgct tcgtatgcag 180 tttcatgatt gtgttgtcaa aggttgtgat gcttccctct tgatcgaccc aacaaccgaa 240 agaccatcag agaaaagcgt cgggcgaaat gcaggggtga gaggtttcga gatcatagat 300 gaagctaaga aagaactcga gcttgtttgc ccgaaaacag tctcttgcgc agacattgta 360 actatagcca ccagagactc gattgcatta gcgggtggtc caaagttcaa agtacgaaca 420 ggaagacgcg atggattaag atcaaaccct agtgatgtta aattgctagg accaacagtt 480 tccgtggcta catcaatcaa agcatttaaa tctataggtt ttaatgtaag caccatggtt 540 gcacttattg gtggtggcca cactgtcggt gttgcacatt gtagtctttt tcaggatagg 600 attaaggatc ctaaaatgga cagcaaactg agagctaagt taaagaagtc atgtcgtggc 660 ccgaacgacc catcagtgtt catggaccaa aatacgccat ttagagtaga caatgaaatc 720 tatagacaga tgatacagca acgagcaatc cttagaattg atgacaactt gattcgcgat 780 ggatcaacca ggtcgattgt gtcagatttt gcatacaaca ataaattatt caaagagagt 840 tttgccgaag cgatgcagaa aatgggtgaa ataggagttc ttacgggaga ttctggagag 900 atcaggacaa actgcagagc cttcaacaac tga 933 <210> 3 <211> 990 <212> DNA <213> Arabidopsis <400> 3 atggctcgct tcgatattgt tctactcata ggtctttgcc tcataatctc tgtctttccc 60 gatacaacca ccgcacaact aagccgcggt ttctactcca aaacttgccc caatgtcgaa 120 caaatcgtga gaaatgctgt ccaaaagaaa ataaagaaga cattcgtcgc cgtccctgcc 180 actctccgtc tcttcttcca cgactgcttt gtcaatggtt gtgatgcatc tgtcatgatc 240 caatcaacgc ctaagaacaa ggcagagaag gatcatccag ataacatttc gctagctgga 300 gatggatttg atgtggtgat ccaagctaag aaagcccttg actctaaccc tagttgccgg 360 aacaaggtct catgtgccga tattcttact ttggccaccc gagatgttgt tgttgcggct 420 ggaggaccgt cctatgaagt tgaacttggt aggttcgatg gtttggtatc aaccgcgtca 480 agtgtcgaag ggaacttacc aggaccttcg gacaatgtag acaaacttaa tgctcttttc 540 acgaaaaaca aacttaccca agaagatatg attgctcttt cagcggctca cacccttgga 600 ttcgcacatt gtggcaaggt ctttaaaaga atccacaaat tcaacggcat caactccgtg 660 gacccaactt taaacaaggc ttacgctatt gagcttcaaa aggcttgtcc taagaatgtg 720 gacccaagaa ttgcaatcaa catggaccca gtcacgccca agacgtttga caacacttac 780 ttcaagaatc ttcaacaagg caaaggtctc ttcacctccg atcaagttct cttcacggat 840 ggtcgctcta ggcccaccgt taatgcttgg gcctcaaact ctacggcctt taaccgcgct 900 tttgtgatag ccatgaccaa acttggccgt gttggagtta agaatagcag caatggaaac 960 atccgtcgtg attgtggtgc gtttaactag 990 <210> 4 <211> 990 <212> DNA <213> Arabidopsis <400> 4 atggcgcggt tcagtctggt tgtagtcgtg actcttagtc ttgccatctc tatgttccct 60 gacacaacca ctgctcaact caaaaccaat ttctacggaa actcatgtcc gaatgttgaa 120 caaatcgtga aaaaagtcgt ccaagaaaaa atcaaacaga ccttcgtcac catcccagct 180 actctccgcc tcttcttcca cgattgcttc gtcaatggat gtgatgcgtc ggtcatgatt 240 caatcaacgc ctaccaacaa agccgagaag gatcatccag acaatatttc tttggccgga 300 gatggatttg acgttgtgat caaagccaag aaagctcttg acgctatccc aagttgcaaa 360 aacaaagtct cttgtgctga tattcttgct ttagccaccc gtgatgttgt tgtcgccgcc 420 aaaggtccgt cgtacgcagt ggaactcgga aggtttgatg gtttggtgtc gactgcggct 480 agcgttaacg gaaacttgcc cggaccaaat aacaaagtta cagaacttaa caagcttttt 540 gccaaaaaca aacttaccca agaggacatg atcgctcttt cagcggctca cacacttgga 600 ttcgcccatt gtggcaaagt gttcaacaga atctacaact tcaacctcac acacgccgtt 660 gacccaactc taaacaaagc ctacgctaaa gaacttcagt tggcttgtcc caagacagtt 720 gacccaagaa tcgccatcaa catggaccca accactccaa gacaattcga caacatttac 780 ttcaagaatc tgcaacaagg caaaggactc ttcacttccg accaagttct cttcaccgat 840 ggtcgctcaa agcccaccgt taacgattgg gccaagaatt ctgttgcttt caacaaggct 900 gt; attcgtcgtg actgtggtgc ctttaactga 990

Claims (6)

A peroxidase gene having the nucleotide sequence of SEQ ID NO: 1.
A peroxidase gene having the nucleotide sequence of SEQ ID NO: 2.
A peroxidase gene having the nucleotide sequence of SEQ ID NO: 3.
A peroxidase gene having the nucleotide sequence of SEQ ID NO: 4.
6. A vector comprising an RNA interference (RNAi) cassette for any one of the peroxidase genes selected from claims 1 to 4.
A transformant into which the vector of claim 5 has been introduced.

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