KR101251723B1 - Transformed Plants having increased campesterol contents - Google Patents

Abstract

The present invention provides a transgenic plant with increased camphorsterol content. The transgenic plant of the present invention has a high content of camphorsterol and can be used for the preparation of food or medicine for lowering cholesterol levels or preventing or treating coronary heart disease, and exhibits a dwarf phenotype, which is suitable for landscaping and differentiation.

Description

Transformed Plants having increased campesterol contents}

The present invention relates to a transgenic plant having an increased camphorsterol content.

Campesterol is a plant sterol found in many plant systems and is one of phytosterols having a chemical structure similar to cholesterol. It is different from cholesterol in that methyl group is located at carbon position 24 of the side chain, and it is classified as 4-desmethylsterol of cholestane family. It is included in vegetables, fruits and nuts, but in low concentrations.

Campestrol is known to play a role in reducing the risk of coronary heart disease by lowering total-cholesterol and LDL-cholesterol levels. It is known to inhibit dietary cholesterol absorption and endogenous cholesterol resorption of the gastrointestinal tract, to excrete cholesterol into feces to reduce serum cholesterol levels, to replace cholesterol with bile salts or to lower cholesterol levels by inhibiting cholesterol esterification in the intestinal mucosa. .

Brassinosteroids are plant steroid hormones that affect a variety of physiological processes, including embryogenicity, cell growth, vasculature, reproduction and aging. Brassinosteroids, including brassinolide and castasterone, are synthesized from camphorster through a series of enzymatic actions including reduction, hydroxylation, epimerization and oxidation.

Sterol methyl transferase 2 (STEROL METHYLTRANSFERASE 2; SMT2 ), DE-ETIOLATED2 ( DET2 ) and DWARF4 ( DWF4 ) are enzymes involved in brassinosteroid biosynthesis in Arabidopsis thaliana.

The SMT2 is known to play an important role in adjusting the ratio of the C28 parameters and sterol methyl transferase, C28 sterols (campesterol) and C29 sterols (sitosterol).

The DET2 mediates sterol 5α-reductase, and (24R)-of (24R) -24-methylcholest-4-en-3one ((24R) -24-methylcholest-4-en-3-one) It is known to catalyze the reduction to 24-methyl-5a-cholestan-3-one ((24R) -24-methyl-5a-cholestan-3-one).

It mediates the DWF4 sterol C22α-hydroxylase and catalyzes several C-22 hydroxylations, which are known to be important rate-determination steps in the brassinosteroid biosynthetic pathway.

Accordingly, the present inventors have studied the brassinosteroid biosynthetic pathway, and as a result, prepared a transgenic plant with significantly increased camphorsterol content compared to the wild type and completed the present invention.

The present invention is to provide a transgenic plant with increased camphorsterol content.

The present invention also provides a method for producing a transgenic plant with increased camphorsterol content.

As a means for solving the above problems, the present invention provides RNA interference recombination vector for det2, dwf4 and smt2 gene.

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

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

The present invention also provides a method of increasing the amount of camphorsterol in a plant using the recombinant vector.

The present invention also provides a method for producing camphorsterol using the transgenic plant.

The present invention also provides a plant extract containing camphorsterol extracted from the transgenic plant.

Transgenic plants of the invention have an increased camphorsterol content. Therefore, it can be used for the manufacture of food or medicine for lowering cholesterol level or preventing or treating coronary heart disease. In addition, since the transgenic plant of the present invention exhibits a dwarf phenotype, it uses less water, is resistant to wind and rainfall damage, is advantageous for making seeds or fruits, is easier to handle and collect, and has better doping resistance. . Moreover, it is suitable not only for flower beds or cut flowers, but also for landscaping and differentiation.

Figure 1A shows a schematic diagram of the RNA interference recombinant vector according to the present invention, Figure 1B is a diagram showing the amplification region in the transcription product of each gene for the vector (black band: exon sequence, red band: the sequence of the example).
Figure 2A is a diagram showing the phenotype of wild-type plants and transformed plants (T3), Figure 2B is a diagram confirming knock-down mutations in the transformed plant (T3) by semi-quantitative RT-PCR analysis. (No. 1 in the figure: wild type plants; no. 2, 3: weak phenotype det2: dwf4: smt2 RNA interfering transgenic plants; no. 4, 5: strong phenotype det2: dwf4: smt2 RNA interfering transgenic plants)
Figure 3A is a diagram showing the phenotype of wild-type plants and transformed plants (T4), Figure 3B is a diagram showing the knockdown mutations in the transformed plant (T4) through real-time RT-PCR analysis. (Black bars: wild type; oblique, spotted bars: strong phenotype det2: dwf4: smt2 interference transgenic plants)

The present invention relates to a RNA interference recombinant vector for the det2, dwf4, and smt2 genes, a microorganism transformed with the recombinant vector, and a plant transformed with the recombinant vector, and a method for producing camphorsterol using the transformed plant. will be.

Hereinafter, the present invention will be described in more detail.

The present invention relates to RNA interference recombinant vectors for det2 (De-Etilolated 2), dwf4 (dwarf 4), and smt2 (sterol methyltransferase 2) genes.

RNA interference recombinant vectors of the present invention include the sense sequences for RNA interference of the det2, dwf4, and smt2 genes mentioned below, and their respective antisense sequences.

Sequence for RNA interference of the det2 gene contained in the RNA interference recombinant vector of the present invention is a part of the DET2 coding sequence (GenBank accession no. U53860) described in SEQ ID NO: 1 (sense base), that is, the base described in SEQ ID NO: 1 DNA of about 100 to about 500 bp of the sequence may be used, and preferably, a DNA consisting of the nucleotide sequence of SEQ ID NO: 3 may be used. Also included within the scope of the present invention are variants of the nucleotide sequence of SEQ ID NO: 3 which can be expressed in the transformed plant and bind to det2 mRNA to inhibit DET2 expression.

Sequence for RNA interference of the dwf4 gene included in the RNA interference recombinant vector of the present invention is a part of the DWF4 coding sequence (GenBank accession no. NM_114926) described in SEQ ID NO: 4, that is, the base described in SEQ ID NO: 4 About 100 to about 500 bp of DNA may be used in the sequence, and preferably, a DNA consisting of the nucleotide sequence of SEQ ID NO: 6 may be used. Also included within the scope of the present invention are variants of the nucleotide sequence of SEQ ID NO: 6 which can be expressed in the transformed plant and bind to dwf4 mRNA to inhibit DWF4 expression.

Sequence for RNA interference of the smt2 gene included in the RNA interference recombinant vector of the present invention is a part (Sense base) of the SMT2 coding sequence (GenBank accession no. NM_101884) described in SEQ ID NO: 7, that is, the base described in SEQ ID NO: 7 DNA of about 100 to about 500 bp of the sequence may be used, and preferably, a DNA consisting of the nucleotide sequence of SEQ ID NO: 9 may be used. Also included within the scope of the present invention are variants of the nucleotide sequence of SEQ ID NO: 7 that are expressed in the transformed plant and can bind to smt2 mRNA and inhibit the expression of SMT2.

The antisense base sequence means a complementary sequence to the sense base sequence.

The abovementioned variants include base sequences each having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the corresponding base sequence, and corresponding Allelic or interspecies variants of the nucleotide sequence, or fragments thereof. The "% sequence homology" for polynucleotides and polypeptides is determined by comparing two optimally arranged sequences with a comparison region, wherein a portion of the polynucleotide and polypeptide sequences in the comparison region are the reference sequences for the optimal alignment of the two sequences. It may include additions or deletions (ie gaps) compared to (not including additions or deletions).

The vector usable in the present invention is a vector for plant expression.

"Vector" refers to DNA fragment (s), nucleic acid molecules that are delivered into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. "Expression vector" means 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 eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The plant expression vector used in the present invention is an RNA interference recombinant vector, which is a recombinant vector designed to generate a stem looped dsRNA that induces RNA interference on det2, dwf4 and smt2 genes in the transformed plant. A “stem loop” refers to a structure composed of a portion of a double chain (stem) generated by hydrogen bonding between reverse repeat sequences present on a single chain RNA and a loop portion therebetween, also called a hairpin loop. Therefore, the plant expression vector used in the present invention comprises a sense sequence (or antisense) encoding a sense (or antisense) RNA of a region of mRNA for DET2 , DWF4 , SMT2 , and a sequence complementary to the present sense sequence, a spacer -Includes a DNA having a structure in which the two sequences are arranged opposite each other with a region interposed therebetween and operably linked to the promoter. Here, the DNA constituting the spacer region is usually 1 to 10,000 bases, although the length thereof is not particularly limited as long as the repeating sequence adjacent thereto can hydrogen bond. The base sequence of the DNA constituting the spacer region can be any sequence without particular limitation, and preferably, a 1,352 bp intron obtained from the CHSA gene (penunia chalcone synthase A) (hereinafter referred to as 'CHSA intron') can be used. .

Plant expression vectors of the invention also include selection markers, promoters and terminators.

Markers usable in the present invention are typically nucleic acid sequences having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as glyphosate, herbicide resistance genes such as phosphinothricin, kanamycin, G418, bleomycin, hygromycin, chloramphenicol It doesn't happen. Preferably the Basta resistance (BAR) gene is used.

Promoters usable in the present invention include promoters capable of initiating transcription in plant cells, including CaMV35S, actin, ubiquitin, pEMU, histone promoters, and the like, and preferably CaMV35S promoters.

Terminators usable in the present invention may use conventional terminators, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (agrobacterium tumefaciens) Terminator of the octopine gene, and the rrnB1 / B2 terminator of Escherichia coli, and the like. An octopine synthase (OCS) terminator is preferably used.

In particular, a preferred RNA interference recombinant vector in the present invention comprises a BAR gene, a CaMV35S promoter, an octopine synthase terminator and a CHSA intron having herbicide resistance between two T-DNA borders, and RNA interference of det2, dwf4 and smt2 genes. The sense sequences for and the antisense sequences for RNA interference of the det2, dwf4 and smt2 genes are plant expression recombinant vectors arranged to be reversed from each other with CHSA interposed therebetween and operably linked to the promoter. The vector according to an embodiment of the present invention is a pFGC5941- DET2 : DWF4 : SMT2 vector described in FIG. 1A.

The sense sequences for RNA interference of the det2 gene, dwf4 gene, and smt2 gene included in the RNA interference recombinant vector of the present invention, and their antisense sequences are in any order as long as the transcription product can form a stable stem loop structure. Can be arranged in a det2: dwf4: smt2 order from the N-terminal side.

As used herein, operably linked genes and promoters refer to conditions in which nucleic acid sequences are linked to perform their intended function. For example, operably linking a promoter sequence to a desired nucleotide sequence refers to linking the promoter sequence to the desired nucleotide sequence in a manner that enables the promoter sequence to direct transcription of the desired nucleotide sequence.

RNA interfering recombinant vectors of the invention can be introduced into cultured host cells using well known techniques such as infection, transduction, transfection, electroporation and transformation. Representative examples of hosts include bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; And plant cells. Suitable culture media and conditions for host cells are known in the art. In the present invention, it is preferable to use the strain Agrobacterium for use in plant transformation.

Transformation of a plant by the RNA interference recombinant vector of the present invention may be performed by any method for transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants.

For example, calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) , Electroporation of protoplasts (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179 -185), Agrobacterium (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), invasion of plants or transformation of mature pollen or vesicles Tumefaciens ( Agrobacterium) tumefaciens ) mediated gene transfer ( incomplete ) infection with virus (EP 0 301 316) and the like can be suitably selected. Preferably Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

Optimal procedures for the transformation of plants with Agrobacterium vectors vary depending on the type of plant to be transformed. Exemplary methods for Agrobacterium mediated transformation include transformation of explants of embryonic axis, shoot tip, stem or leaf tissue, derived from sterile seedlings and / or small plants. Such transformed plants can be reproduced by sexual reproduction or by cell or tissue culture. Agrobacterium mediated transformation has previously been described for many different kinds of plants and methods for such transformation can be found in literature known in the art.

Agrobacterium strains for plant transformation that can be used in the present invention can be used any strains commonly used. Specifically, in the present invention, Agrobacterium tumefaciens, which is known to be highly pathogenic to penetrate plant samples and deliver foreign genes, and more preferably, a GV3101 strain containing a hygromycin resistance gene pCAMBIA 1300 plasmid Agrobacterium tumefaciens can be used.

Plants to be transformed in the present invention can be used for plants that can be used economically, such as Riatris, Echinacea, Gaura, Phlox as well as the Arabidopsis used in the present embodiment. Plants transformed with the RNA interference recombinant vector of the present invention exhibit increased camphorsterol content and dwarf phenotype.

The present invention also relates to a method for increasing the content of camphorsterol in a plant, including transformation with the RNA recombinant vector.

In addition, the present invention relates to a method for producing camphorsterol, characterized in that the extraction from the plant transformed with the RNA recombinant vector.

The present invention also relates to a plant extract extracted from the transgenic plant.

Extraction of camphorsterol from the transgenic plant of the present invention can be carried out by conventional methods known in the art for the extraction of phytosterols.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to illustrate the invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

Example  One. RNA  Interference Recombinant Vector Fabrication

(One) DET2 , DWF4 , SMT2  Battlefield cDNA Cloning

Arabidopsis DET2 (GenBank accession no. U53860), DWF4 (GenBank accession no. NM_114926) and SMT2 using RT-PCR Full length cDNA of GenBank accession no. NM_101884 was obtained from Arabidopsis leaves and cloned into pGEM-T (Promega, USA) to obtain pGEM-T- DET2 , pGEM-T- DWF4 and pGEM-T- SMT2 . Prepared.

Specifically, after obtaining the total RNA from the Arabidopsis leaf using the RNeasy Plant Mini Kit (Qiagen, USA), cDNA was secured using the ImProm-II ^TM Reverse Transcription System (Promega). After reacting 1 μg of total RNA with oligo-dt primer at 70 ° C. for 5 minutes, ImProm-II ^TM 5X reaction buffer, 2.5 mM MgCl ₂ , 0.5 mM dNTPs Mix and ImProm-II ^TM reverse transcriptase were reacted at 25 ° C. After reacting for 5 minutes, the mixture was reacted at 42 ° C. for 1 hour, and then reacted at 70 ° C. for 15 minutes to obtain cDNA. PCR was performed with the obtained cDNA as a template. In PCR, the amplification program was initially 94 ° C. 5 minutes, followed by 94 ° C. 1 minute; 55 ° C. 1 minute; 30 cycles of 72 ° C. per minute, and final extension of 72 ° C. for 10 minutes. The sense and antisense primer sequences used for PCR are as follows.

det2 sense and antisense primer

5'-ATGGAAGAAATCGCCGATAAAAC-3 '(SEQ ID NO: 10)

5'-TCAGTACACAAAAGGAATAACAG-3 '(SEQ ID NO: 11)

dwf4 sense and antisense primer

5'-ATGTTCGAAACAGAGCATC-3 '(SEQ ID NO: 12)

5'-TTACAGAATACGAGAAACCC-3 '(SEQ ID NO: 13)

smt2 sense and antisense primer

5'-ATGGACTCTTTAACACTCTTC-3 '(SEQ ID NO: 14)

5'-TCAAGAACTCTCCTCCGGTG-3 '(SEQ ID NO: 15)

Each of the amplified genes was ligated to the pGEM-T vector (Promega). 3 μl of each amplified gene, 1 μl of pGEM-T vector and 1 μl of T4 DNA ligase were reacted at 25 ° C. for 2 hours.

(2) pFGC5941 - DET2 : DWF4 : SMT2  Recombinant vector production

PFGC5941- DET2: DWF4: SMT2 was prepared using the prepared cDNAs of AtDET2, AtDWF4, and AtSMT2 (FIG. 1B).

To prepare pFGC5941- DET2 : DWF4 : SMT2 vectors, det2 455 bp fragments were PCR amplified from pGEM-T- DET2 . The sense and antisense primer sequences are as follows, respectively.

5'-AAGCTTTCATTTACCCTCTTCG-3 '(SEQ ID NO: 16)

5'-GTCGACGAACTTGGCAATGTAC-3 '(SEQ ID NO: 17)

The amplified det2 fragment was cleaved with HindIII and Sal I, and the cleaved fragment was ligation into pUC18 to generate pUC18- DET2 . The dwf4 361 bp fragment was then amplified using the following primer sequence.

5'-GTCGACGCCGGACATGAGACTTC-3 '(SEQ ID NO: 18)

5'-GGTACCGCCATCTCCAAGGATTAA-3 '(SEQ ID NO: 19)

By cutting the amplified fragment dwf4 the Sal I and Kpn I, and ligated to the cut sections to the pUC18- DET2, pUC18- DET2: was prepared DWF4.

SMT2 289 bp fragment was amplified using the following primer sequence.

5'-GGTACCCTAGACGTCGGATGCGGT-3 '(SEQ ID NO: 20)

5'-GAATTCCATAGATCCGGGTTTCAA-3 '(SEQ ID NO: 21)

Ligated to DWF4 pUC18- DET2:: cut the amplified fragment smt2 the KpnI and EcoRI, and the truncated fragment was prepared pUC18- DET2 SMT2: DWF4.

PCR products of DET2 : DWF4 : SMT2 were obtained using the following primer sequences.

5'-TCTAGAGGCGCGCCTCATTTACCCTCTTCGC-3 '(SEQ ID NO: 22)

5'-GGATCCATTTAAATCATAGATCCGGGTTTCAA-3 '(SEQ ID NO: 23)

The resulting PCR product was subjected to sense and antisense directions using the AscISwaI and BamHIXbaI restriction enzyme sites of the pFGC5941 vector (binary vector (http://www.chromdb.org/rnai/vector_info.html) for the production of double stranded RNA in plants). orientation was inserted above and below the chalcone synthase intron.

pFGC5941- DET2 : DWF4 : SMT2 The directionality of the insertion into the vector and the suitability of the reading frame were confirmed by DNA sequencing.

Example  2. Preparation of Transgenic Strains

Agrobacterium tumefaciens GV3101 cells comprising pFGC5941- DET2 : DWF4 : SMT2 vectors were prepared by electroporation for the preparation of recombinant Agrobacterium tumefaciens to be used for plant transformation.

Specifically, 1 μl (50ng / μl or more) of pFGC5941-DET2: DWF4: SMT2 recombinant vector DNA prepared above was added to 200 μl of Agrobacterium competent cells (GV3101), and left on ice for 30 minutes, followed by alcohol sterilization. Put it in the micro-electroporation chamber (Micro-Electroporation Chamber) using a current of 2.4kV was transferred to ice.

Into this Agrobacterium solution, 800 μl of LB medium was added to a 15 ml tube using a pipette, grown at 25 ° C. for 4 hours, and then in an LB solid medium containing 50 mg / L gentamicin and 50 mg / L kanamycin antibiotic. The plate was incubated at 25 ° C. for 2 days.

Cultured Agrobacterium single colony was liquid cultured, plasmid DNA was extracted and confirmed by PCR method. PCR conditions were performed for 5 minutes at 94 ° C total denaturation, 30 ° C denaturation at 94 ° C, 30 ° C annealing at 55 ° C, 30 ° elongation at 72 ° C, 7 minutes at extension 72 ° C, and a total of 30 cycles. The DNA having the nucleotide sequence of SEQ ID NO: 3 was inserted into the selected Agrobacterium clone and used for plant transformation.

Example  3. Preparation of Transgenic Plants

(1) Infection with Agrobacterium

After spin down of the prepared transformed Agrobacterium, resuspended in OD ₆₀₀ = 0.8 in 10% sucrose solution, Silwet L-77 (Cat. No. VIS-02, Lehle Seed, USA) at 0.05% concentration. Was added and mixed well. After seeding, the solution was sprayed onto the Arabidopsis every 3 times every other day on the Arabidopsis, which rose after the rosette generation. The Arabidopsis germinated in soil, and was grown in a photoperiod 16h, 22 ℃ growth chamber.

(2) Selection of Transgenic Plants

Seedlings with main leaves after sowing presumed to be transgenic plants and non-transgenic plants were sprayed with 0.4% BASTA (Bayer, Germany) every other week for one week and the surviving seedlings were potted. Transformant seeds were obtained. After germination, the transformed plants were reselected using 0.4% BASTA in the same manner as above. Culture conditions were photoperiod 16h, 22 ℃.

This confirms that the knock-down mutants of the present inventors can exhibit excellent seed productivity, unlike the loss-of-function mutant or knockout mutants, which show a significant reduction in reproduction. Thus, it can be seen that it has a commercial advantage. This seed productivity seems to be due to gene expression remaining in RNAi plants.

Experimental Example  1. Wild type plants and DET2 : DWF4 : SMT2 RNA  Comparison of Phenotypes of Interfering Transgenic Plants

(One) T3  Phenotype Comparison of Generation Transgenic Plants

RNAi knock-down plant lines were selected from the T3 generation by isolation analysis using selective antibiotic markers. After seeding, the leaves were sprayed every other day for 1 week with 0.4% BASTA, and the culture conditions were grown for 4 weeks at 22 ° C. of 16 h of photoperiod and observed the phenotype. Morphological differences were observed between wild type plants and transgenic plants. (FIG. 2A) Transgenic plants showed a clear change in phenotype. Transgenic plants corresponding to strong and weak inhibition showed dwarf and quasi-dwarf phenotypes, respectively.

(2) T4  Phenotype Comparison of Generation Transgenic Plants

BASTA-resistant T4 plants were harvested from 4 and 5 dwarf strains as shown in FIG. 2A to investigate if the properties shown in the T3 generation were inherited in the next generation. As above, when seedlings emerged after seeding, they were sprayed every other day for 1 week with 0.4% BASTA, and the culture conditions were observed for 4 weeks at 22 ° C. of photoperiod 16h. The transgenic plants showed a clear modified phenotype (FIG. 3A). On the other hand, the results of morphometric analysis of the transformed plant was as Table 1 below.

Table 1 Morphometric Analysis of Wild-type and T4 Transgenic Plants

As shown in Table 1 above, the transgenic plant had short flowering height, shorter flanks and shorter petioles.

Experimental Example  2. RNA  Isolation and Expression Pattern Confirmation

(One) T3  Generation of transgenic plants RNA  Separation and semi-quantitative RT - PCR  analysis

Semi-quantitative RT PCR analysis confirmed the transcription levels of the DET2, DWF4, SMT2 and CYP85A1 genes using RNA isolated from each transformant strain.

Specifically, using the RNeasy Plant Mini Kit (Qiagen, USA), total RNA was isolated from the T3 generation Arabidopsis leaf obtained in Experimental Example 1. (1) above.

The isolated RNA was reverse transcribed using DNaseI (Takara, Japan) -treated total RNA using ImProm-II ^™ Reverse Transcription System (Promega, USA) and oligo-dT primer according to the manufacturer's instructions. The first strand cDNA was then PCR amplified using DET2, DWF4, SMT2 and CYP85A1 gene-specific primers and internal standard 18S rRNA primers.

A 780 bp fragment was prepared by amplifying the DET2 gene with the following primer sets.

5'-ATGGAAGAAATCGCCGATAAAACC-3 '(SEQ ID NO: 24)

5'-ACACAAAAGGAATAACAGCTTTAC-3 '(SEQ ID NO: 25)

The 1,540 bp fragment was prepared by amplifying the DWF4 gene with the following primer sets.

5'-ATGTTCGAAACAGAGCATCATAC-3 '(SEQ ID NO: 26)

5'-GAATACGAGAAACCCTAATAGGC-3 '(SEQ ID NO: 27)

The SMT2 gene was amplified with the following primer sets to prepare 1,080 bp fragments.

5'-ATGGACTCTTTAACACTCTTC-3 '(SEQ ID NO: 28)

5'-AGAACTCTCCTCCGGTGACTC-3 '(SEQ ID NO: 29)

The 1,150 bp fragment was prepared by amplifying the CYP85A1 gene with the following primer sets.

5'-GCTCGCTTCTGTCTCTCA-3 '(SEQ ID NO: 30)

5'-TCATCCCCTCCTATTTCC-3 '(SEQ ID NO: 31)

Internal standard 18S rRNA primers used the following set.

5'-GGATGGGTCGGCCGGTC-3 '(SEQ ID NO: 32)

5'-CAGGCTGAGGTCTCGTTC-3 '(SEQ ID NO: 33)

In PCR, the amplification program was initially 94 ° C. 5 minutes, then 94 ° C. 1 minute; 55 ° C. 1 minute; 28-35 cycles of 72 ° C. per minute, and final extension of 72 ° C. for 10 minutes. The experiment was repeated three more times to obtain similar results.

The results of RT-PCR analysis are shown in Figure 2B. As shown in Figure 2B, it was confirmed that the mRNA levels of the DET2, DWF4, SMT2 gene in the transgenic plant was reduced compared to the level of wild type. The knockdown of gene expression was classified into strong and weak inhibitory groups. This indicates dose-dependent dwarfism.

On the other hand, unlike the transcription levels of DET2, DWF4 and SMT2, CYP85A1 was up-regulated in transgenic plants. This is consistent with the feedback regulatory mechanism of the brassinosteroid biosynthesis gene, with reduced brassinosteroid biosynthesis or erroneous responses de-inhibiting the expression of the biosynthetic gene, including CYP85A1. Thus, the increased transcription level of CYP85A1 in the transgenic plants of the present invention suggests that the overall biosynthetic activity and the subsequent signal response are down regulated due to RNA interference effects by transformation.

(2) T4  Generation of transgenic plants RNA Separation and Real Time Quantitation RT - PCR  analysis

CDNA was obtained from the T4 generation Arabidopsis leaves obtained in Experimental Example 1. (2) above in the same manner as in Example 1. (1) above. Reverse transcription of DNase I (Takara, Japan) -treated total RNA was performed using oligo-dT primers and a First-Strand cDNA Synthesis kit (Roche Diagnostics GmbH, Germany). For quantitative real-time PCR, gene-specific PCR primers and fluorescence probes for Taq-Man assays were designed by Assays-by-Design Service (Applied Biosystems, USA). Gene expression was analyzed using TaqMan Universal PCR Master Mix and 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Amplification conditions were an initial cycle of 50 ° C. for 2 minutes, one cycle of 95 ° C. for 10 minutes, followed by 95 cycles of 15 seconds and 60 cycles of 1 cycle of 40 minutes.

In the analysis of transgenic plants, the gene-specific primers and probes used for quantitative real-time PCR are as follows:

[For DET2]

Forward: 5'-TTTGGAGAGGCGATTGAGTG-3 '(SEQ ID NO. 34)

Reverse: 5'-CTCTTCCTTGAACTTGGCAATG-3 '(SEQ ID NO: 35)

DET2-probe

FAM-TGGGCTGTTATGACTTGGTCTTGGG-NFQ;

[For DWF4]

Forward: 5'-CAGAGGATGAAGCAGAGATGAG-3 '(SEQ ID NO: 36)

Reverse: 5'-TGAGATCGAGAATTTGCTCCG-3 '(SEQ ID NO: 37)

DWF4-Probe

FAM-ACAGACGATGATCTTTTGGGATGGGTT-NFQ;

[For SMT2]

Forward: 5'-CCAAAAGAAATCGAAACCGCC-3 '(SEQ ID NO: 38)

Reverse: 5'-GCGTCTTTGTGAGATTTTCCG-3 '(SEQ ID NO: 39)

SMT2-Probe

FAM-TGTCCCCATCCCCACTCGTATATGT-NFQ;

[For CYP85A1]

Forward: 5'-ACAGAGCAGAAAACAGAGTGAG-3 '(SEQ ID NO: 40)

Reverse: 5'-GAAGGAGAGCGGAACAGAG-3 '(SEQ ID NO: 41)

CYP85A1-Probe

FAM-TGGGAGCAATGATGGTGATGATGGG-NFQ;

[For 18S rRNA]

Pre-designed primers and probes

(Taqman assay-on-demand gene expression products, Applied Biosystems).

Amplification of 18S rRNA was used for standardization of real time PCR results.

Relative expression levels of each gene as a result of RT-PCR analysis are shown in FIG. 3B. As shown in FIG. 3B, it was confirmed that mRNA levels of DET2, DWF4, and SMT2 genes in the transgenic plants were reduced compared to those of the wild type. That is, it was confirmed that mRNA levels of DET2, DWF4, and SMT2 genes were down-regulated by about 20, 56, and 52% from transgenic plants. On the other hand, the expression of the CYP85A1 gene was confirmed to be up-regulated by up to 27%.

Experimental Example  3. Sterol and Brassinosteroid  Profile analysis

20 g of the stem, flowers, leaves, and long-gap stems of the T4 generation cephalopod obtained in Experimental Example 1. (2) above were harvested to purify and quantify sterols and brassinosteroids. The purification and quantification was performed according to the method described in Fujioka et al., Plant Physiology 2002 Oct; 130 (2): 930-9, An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway. The analysis results are shown in Table 2 below.

Table 2. Endogenous Sterol and Brassinosteroid Levels of Wild-type and T4 Transgenic Plants

As shown in Table 2 above, the camphorsterol content of the triple knockdown transgenic plant was 186 μg / g FW. This content is 4.2 times higher than the level in wild type (44.2 μg / g FW). Phytosterols, including camphorsterol, are very important for human health, and in particular, camphorsterol is widely known to exhibit cholesterol-lowering and anticancer effects.

On the other hand, cytosterol and stigmasterol contents decreased by 35 and 31% of wild type levels, respectively. In transgenic plants, 6-dioxocatasterone (6-deoxocathasterone: DeoxoCT), 6-dioxoteasterone (6-deoxoteasterone: 6-DeoxoTE), 3-dihydro-6-dioxoteasterone ( 3-dehydro-6-deoxoteasterone: 6-Deoxo3DT), 6-dioxotyphasterol (6-deoxotyphasterol: 6-DeoxoTY), 6-dioxocastasterone (6-deoxocastasterone: 6-DeoxoCS), typhasterol : TY), and castasterone (CS) levels were reduced by 19, 50, 15, 43, 19, 38 and 54% of the levels in wild type plants, respectively. Cathasterone (CT), teasterone (TE), 3-dehydroteasterone (3-DT) and brassinolide (BL) are found in wild-type or transgenic plants. It was not detected.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Transformed plants having increased campesterol contents <130> P10-019-KHU <160> 41 <170> Kopatentin 1.71 <210> 1 <211> 789 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS (222) (1) .. (786) <223> DET2 (Genbank accession no.U53860) <400> 1 atg gaa gaa atc gcc gat aaa acc ttc ttc cga tac tgt ctc ctc act 48 Met Glu Glu Ile Ala Asp Lys Thr Phe Phe Arg Tyr Cys Leu Leu Thr   1 5 10 15 ctt att ttc gcc ggc cca cca acc gcc gtc ctt ctg aaa ttc ctc caa 96 Leu Ile Phe Ala Gly Pro Pro Thr Ala Val Leu Leu Lys Phe Leu Gln              20 25 30 gct cct tac ggt aaa cac aac cgt acc gga tgg ggt ccc acc gta tct 144 Ala Pro Tyr Gly Lys His Asn Arg Thr Gly Trp Gly Pro Thr Val Ser          35 40 45 cca ccg att gct tgg ttc gtc atg gag agc cca acc ttg tgg ctc act 192 Pro Pro Ile Ala Trp Phe Val Met Glu Ser Pro Thr Leu Trp Leu Thr      50 55 60 ctc ctc ctc ttc ccc ttt ggt cgt cac gct ctc aac cct aaa tct cta 240 Leu Leu Leu Phe Pro Phe Gly Arg His Ala Leu Asn Pro Lys Ser Leu  65 70 75 80 ctt cta ttc tct cct tat ctc att cat tac ttc cac cgc acc atc att 288 Leu Leu Phe Ser Pro Tyr Leu Ile His Tyr Phe His Arg Thr Ile Ile                  85 90 95 tac cct ctt cgc ctc ttc cgc agc tcc ttc ccc gcc ggt aaa aac gga 336 Tyr Pro Leu Arg Leu Phe Arg Ser Ser Phe Pro Ala Gly Lys Asn Gly             100 105 110 ttt ccg atc acc atc gcc gcc ttg gct ttc acc ttt aat ctc ctc aat 384 Phe Pro Ile Thr Ile Ala Ala Leu Ala Phe Thr Phe Asn Leu Leu Asn         115 120 125 ggt tat atc cag gcg agg tgg gtt tcg cat tac aag gat gac tac gaa 432 Gly Tyr Ile Gln Ala Arg Trp Val Ser His Tyr Lys Asp Asp Tyr Glu     130 135 140 gac gga aac tgg ttc tgg tgg cgg ttt gtt atc ggt atg gtg gtt ttc 480 Asp Gly Asn Trp Phe Trp Trp Arg Phe Val Ile Gly Met Val Val Phe 145 150 155 160 ata acc ggc atg tat ata aat atc acg tcg gac cgc act ttg gta cga 528 Ile Thr Gly Met Tyr Ile Asn Ile Thr Ser Asp Arg Thr Leu Val Arg                 165 170 175 ttg aag aaa gag aac cgg gga ggt tat gtg ata ccg aga gga ggc tgg 576 Leu Lys Lys Glu Asn Arg Gly Gly Tyr Val Ile Pro Arg Gly Gly Trp             180 185 190 ttc gag ttg gta agc cgt ccg aat tat ttt gga gag gcg att gag tgg 624 Phe Glu Leu Val Ser Arg Pro Asn Tyr Phe Gly Glu Ala Ile Glu Trp         195 200 205 ttg ggc tgg gct gtt atg act tgg tct tgg gcc ggt att gga ttt ttt 672 Leu Gly Trp Ala Val Met Thr Trp Ser Trp Ala Gly Ile Gly Phe Phe     210 215 220 ctg tac acg tgt tcc aat ttg ttt ccg cgt gca cgt gcg agt cac aag 720 Leu Tyr Thr Cys Ser Asn Leu Phe Pro Arg Ala Arg Ala Ser His Lys 225 230 235 240 tgg tac att gcc aag ttc aag gaa gag tat ccc aag act cgt aaa gct 768 Trp Tyr Ile Ala Lys Phe Lys Glu Glu Tyr Pro Lys Thr Arg Lys Ala                 245 250 255 gtt att cct ttt gtg tac tga 789 Val Ile Pro Phe Val Tyr             260 <210> 2 <211> 262 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Glu Glu Ile Ala Asp Lys Thr Phe Phe Arg Tyr Cys Leu Leu Thr   1 5 10 15 Leu Ile Phe Ala Gly Pro Pro Thr Ala Val Leu Leu Lys Phe Leu Gln              20 25 30 Ala Pro Tyr Gly Lys His Asn Arg Thr Gly Trp Gly Pro Thr Val Ser          35 40 45 Pro Pro Ile Ala Trp Phe Val Met Glu Ser Pro Thr Leu Trp Leu Thr      50 55 60 Leu Leu Leu Phe Pro Phe Gly Arg His Ala Leu Asn Pro Lys Ser Leu  65 70 75 80 Leu Leu Phe Ser Pro Tyr Leu Ile His Tyr Phe His Arg Thr Ile Ile                  85 90 95 Tyr Pro Leu Arg Leu Phe Arg Ser Ser Phe Pro Ala Gly Lys Asn Gly             100 105 110 Phe Pro Ile Thr Ile Ala Ala Leu Ala Phe Thr Phe Asn Leu Leu Asn         115 120 125 Gly Tyr Ile Gln Ala Arg Trp Val Ser His Tyr Lys Asp Asp Tyr Glu     130 135 140 Asp Gly Asn Trp Phe Trp Trp Arg Phe Val Ile Gly Met Val Val Phe 145 150 155 160 Ile Thr Gly Met Tyr Ile Asn Ile Thr Ser Asp Arg Thr Leu Val Arg                 165 170 175 Leu Lys Lys Glu Asn Arg Gly Gly Tyr Val Ile Pro Arg Gly Gly Trp             180 185 190 Phe Glu Leu Val Ser Arg Pro Asn Tyr Phe Gly Glu Ala Ile Glu Trp         195 200 205 Leu Gly Trp Ala Val Met Thr Trp Ser Trp Ala Gly Ile Gly Phe Phe     210 215 220 Leu Tyr Thr Cys Ser Asn Leu Phe Pro Arg Ala Arg Ala Ser His Lys 225 230 235 240 Trp Tyr Ile Ala Lys Phe Lys Glu Glu Tyr Pro Lys Thr Arg Lys Ala                 245 250 255 Val Ile Pro Phe Val Tyr             260 <210> 3 <211> 455 <212> DNA <213> Arabidopsis thaliana <220> <221> misc_feature (222) (1) .. (455) <223> DET2 fragment for RNAi <400> 3 tcatttaccc tcttcgcctc ttccgcagct ccttccccgc cggtaaaaac ggatttccga 60 tcaccatcgc cgccttggct ttcaccttta atctcctcaa tggttatatc caggcgaggt 120 gggtttcgca ttacaaggat gactacgaag acggaaactg gttctggtgg cggtttgtta 180 tcggtatggt ggttttcata accggcatgt atataaatat cacgtcggac cgcactttgg 240 tacgattgaa gaaagagaac cggggaggtt atgtgatacc gagaggaggc tggttcgagt 300 tggtaagccg tccgaattat tttggagagg cgattgagtg gttgggctgg gctgttatga 360 cttggtcttg ggccggtatt ggattttttc tgtacacgtg ttccaatttg tttccgcgtg 420 cacgtgcgag tcacaagtgg tacattgcca agttc 455 <210> 4 <211> 1542 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS (222) (1) .. (1539) <223> DWF4 (GenBank accession no.NM_114926) <400> 4 atg ttc gaa aca gag cat cat act ctc tta cct ctt ctt ctt ctc cca 48 Met Phe Glu Thr Glu His His Thr Leu Leu Pro Leu Leu Leu Leu Pro   1 5 10 15 tcg ctt ttg tct ctt ctt ctc ttc ttg att ctc ttg aag aga aga aat 96 Ser Leu Leu Ser Leu Leu Leu Phe Leu Ile Leu Leu Lys Arg Arg Asn              20 25 30 aga aaa acc aga ttc aat cta cct ccg ggt aaa tcc ggt tgg cca ttt 144 Arg Lys Thr Arg Phe Asn Leu Pro Pro Gly Lys Ser Gly Trp Pro Phe          35 40 45 ctt ggt gaa acc atc ggt tat ctt aaa ccg tac acc gcc aca aca ctc 192 Leu Gly Glu Thr Ile Gly Tyr Leu Lys Pro Tyr Thr Ala Thr Thr Leu      50 55 60 ggt gac ttc atg caa caa cat gtc tcc aag tat ggt aag ata tat aga 240 Gly Asp Phe Met Gln Gln His Val Ser Lys Tyr Gly Lys Ile Tyr Arg  65 70 75 80 tcg aac ttg ttt gga gaa cca acg atc gta tca gct gat gct gga ctt 288 Ser Asn Leu Phe Gly Glu Pro Thr Ile Val Ser Ala Asp Ala Gly Leu                  85 90 95 aat aga ttc ata tta caa aac gaa gga agg ctc ttt gaa tgt agt tat 336 Asn Arg Phe Ile Leu Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr             100 105 110 cct aga agt ata ggt ggg att ctt ggg aaa tgg tcg atg ctt gtt ctt 384 Pro Arg Ser Ile Gly Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu         115 120 125 gtt ggt gac atg cat aga gat atg aga agt atc tcg ctt aac ttc tta 432 Val Gly Asp Met His Arg Asp Met Arg Ser Ile Ser Leu Asn Phe Leu     130 135 140 agt cac gca cgt ctt aga act att cta ctt aaa gat gtt gag aga cat 480 Ser His Ala Arg Leu Arg Thr Ile Leu Leu Lys Asp Val Glu Arg His 145 150 155 160 act ttg ttt gtt ctt gat tct tgg caa caa aac tct att ttc tct gct 528 Thr Leu Phe Val Leu Asp Ser Trp Gln Gln Asn Ser Ile Phe Ser Ala                 165 170 175 caa gac gag gcc aaa aag ttt acg ttt aat cta atg gcg aag cat ata 576 Gln Asp Glu Ala Lys Lys Phe Thr Phe Asn Leu Met Ala Lys His Ile             180 185 190 atg agt atg gat cct gga gaa gaa gaa aca gag caa tta aag aaa gag 624 Met Ser Met Asp Pro Gly Glu Glu Glu Thr Glu Gln Leu Lys Lys Glu         195 200 205 tat gta act ttc atg aaa gga gtt gtc tct gct cct cta aat cta cca 672 Tyr Val Thr Phe Met Lys Gly Val Val Ser Ala Pro Leu Asn Leu Pro     210 215 220 gga act gct tat cat aaa gct ctt cag tca cga gca acg ata ttg aag 720 Gly Thr Ala Tyr His Lys Ala Leu Gln Ser Arg Ala Thr Ile Leu Lys 225 230 235 240 ttc att gag agg aaa atg gaa gag aga aaa ttg gat atc aag gaa gaa 768 Phe Ile Glu Arg Lys Met Glu Glu Arg Lys Leu Asp Ile Lys Glu Glu                 245 250 255 gat caa gaa gaa gaa gaa gtg aaa aca gag gat gaa gca gag atg agt 816 Asp Gln Glu Glu Glu Glu Val Lys Thr Glu Asp Glu Ala Glu Met Ser             260 265 270 aag agt gat cat gtt agg aaa caa aga aca gac gat gat ctt ttg gga 864 Lys Ser Asp His Val Arg Lys Gln Arg Thr Asp Asp Asp Leu Leu Gly         275 280 285 tgg gtt ttg aaa cat tcg aat tta tcg acg gag caa att ctc gat ctc 912 Trp Val Leu Lys His Ser Asn Leu Ser Thr Glu Gln Ile Leu Asp Leu     290 295 300 att ctt agt ttg tta ttt gcc gga cat gag act tct tct gta gcc att 960 Ile Leu Ser Leu Leu Phe Ala Gly His Glu Thr Ser Ser Val Ala Ile 305 310 315 320 gct ctc gct atc ttc ttc ttg caa gct tgc cct aaa gcc gtt gaa gag 1008 Ala Leu Ala Ile Phe Phe Leu Gln Ala Cys Pro Lys Ala Val Glu Glu                 325 330 335 ctt agg gaa gag cat ctt gag atc gcg agg gcc aag aag gaa cta gga 1056 Leu Arg Glu Glu His Leu Glu Ile Ala Arg Ala Lys Lys Glu Leu Gly             340 345 350 gag tca gaa tta aat tgg gat gat tac aag aaa atg gac ttt act caa 1104 Glu Ser Glu Leu Asn Trp Asp Asp Tyr Lys Lys Met Asp Phe Thr Gln         355 360 365 tgt gtt ata aat gaa act ctt cga ttg gga aat gta gtt agg ttt ttg 1152 Cys Val Ile Asn Glu Thr Leu Arg Leu Gly Asn Val Val Arg Phe Leu     370 375 380 cat cgc aaa gca ctc aaa gat gtt cgg tac aaa gga tac gat atc cct 1200 His Arg Lys Ala Leu Lys Asp Val Arg Tyr Lys Gly Tyr Asp Ile Pro 385 390 395 400 agt ggg tgg aaa gtg tta ccg gtg atc tca gcc gta cat ttg gat aat 1248 Ser Gly Trp Lys Val Leu Pro Val Ile Ser Ala Val His Leu Asp Asn                 405 410 415 tct cgt tat gac caa cct aat ctc ttt aat cct tgg aga tgg caa cag 1296 Ser Arg Tyr Asp Gln Pro Asn Leu Phe Asn Pro Trp Arg Trp Gln Gln             420 425 430 caa aac aac gga gcg tca tcc tca gga agt ggt agt ttt tcg acg tgg 1344 Gln Asn Asn Gly Ala Ser Ser Ser Gly Ser Gly Ser Phe Ser Thr Trp         435 440 445 gga aac aac tac atg ccg ttt gga gga ggg cca agg cta tgt gct ggt 1392 Gly Asn Asn Tyr Met Pro Phe Gly Gly Gly Pro Arg Leu Cys Ala Gly     450 455 460 tca gag cta gcc aag tta gaa atg gca gtg ttt att cat cat cta gtt 1440 Ser Glu Leu Ala Lys Leu Glu Met Ala Val Phe Ile His His Leu Val 465 470 475 480 ctt aaa ttc aat tgg gaa tta gca gaa gat gat aaa cca ttt gct ttt 1488 Leu Lys Phe Asn Trp Glu Leu Ala Glu Asp Asp Lys Pro Phe Ala Phe                 485 490 495 cct ttt gtt gat ttt cct aac ggt ttg cct att agg gtt tct cgt att 1536 Pro Phe Val Asp Phe Pro Asn Gly Leu Pro Ile Arg Val Ser Arg Ile             500 505 510 ctg t aa 1542 Leu <210> 5 <211> 513 <212> PRT <213> Arabidopsis thaliana <400> 5 Met Phe Glu Thr Glu His His Thr Leu Leu Pro Leu Leu Leu Leu Pro   1 5 10 15 Ser Leu Leu Ser Leu Leu Leu Phe Leu Ile Leu Leu Lys Arg Arg Asn              20 25 30 Arg Lys Thr Arg Phe Asn Leu Pro Pro Gly Lys Ser Gly Trp Pro Phe          35 40 45 Leu Gly Glu Thr Ile Gly Tyr Leu Lys Pro Tyr Thr Ala Thr Thr Leu      50 55 60 Gly Asp Phe Met Gln Gln His Val Ser Lys Tyr Gly Lys Ile Tyr Arg  65 70 75 80 Ser Asn Leu Phe Gly Glu Pro Thr Ile Val Ser Ala Asp Ala Gly Leu                  85 90 95 Asn Arg Phe Ile Leu Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr             100 105 110 Pro Arg Ser Ile Gly Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu         115 120 125 Val Gly Asp Met His Arg Asp Met Arg Ser Ile Ser Leu Asn Phe Leu     130 135 140 Ser His Ala Arg Leu Arg Thr Ile Leu Leu Lys Asp Val Glu Arg His 145 150 155 160 Thr Leu Phe Val Leu Asp Ser Trp Gln Gln Asn Ser Ile Phe Ser Ala                 165 170 175 Gln Asp Glu Ala Lys Lys Phe Thr Phe Asn Leu Met Ala Lys His Ile             180 185 190 Met Ser Met Asp Pro Gly Glu Glu Glu Thr Glu Gln Leu Lys Lys Glu         195 200 205 Tyr Val Thr Phe Met Lys Gly Val Val Ser Ala Pro Leu Asn Leu Pro     210 215 220 Gly Thr Ala Tyr His Lys Ala Leu Gln Ser Arg Ala Thr Ile Leu Lys 225 230 235 240 Phe Ile Glu Arg Lys Met Glu Glu Arg Lys Leu Asp Ile Lys Glu Glu                 245 250 255 Asp Gln Glu Glu Glu Glu Val Lys Thr Glu Asp Glu Ala Glu Met Ser             260 265 270 Lys Ser Asp His Val Arg Lys Gln Arg Thr Asp Asp Asp Leu Leu Gly         275 280 285 Trp Val Leu Lys His Ser Asn Leu Ser Thr Glu Gln Ile Leu Asp Leu     290 295 300 Ile Leu Ser Leu Leu Phe Ala Gly His Glu Thr Ser Ser Val Ala Ile 305 310 315 320 Ala Leu Ala Ile Phe Phe Leu Gln Ala Cys Pro Lys Ala Val Glu Glu                 325 330 335 Leu Arg Glu Glu His Leu Glu Ile Ala Arg Ala Lys Lys Glu Leu Gly             340 345 350 Glu Ser Glu Leu Asn Trp Asp Asp Tyr Lys Lys Met Asp Phe Thr Gln         355 360 365 Cys Val Ile Asn Glu Thr Leu Arg Leu Gly Asn Val Val Arg Phe Leu     370 375 380 His Arg Lys Ala Leu Lys Asp Val Arg Tyr Lys Gly Tyr Asp Ile Pro 385 390 395 400 Ser Gly Trp Lys Val Leu Pro Val Ile Ser Ala Val His Leu Asp Asn                 405 410 415 Ser Arg Tyr Asp Gln Pro Asn Leu Phe Asn Pro Trp Arg Trp Gln Gln             420 425 430 Gln Asn Asn Gly Ala Ser Ser Ser Gly Ser Gly Ser Phe Ser Thr Trp         435 440 445 Gly Asn Asn Tyr Met Pro Phe Gly Gly Gly Pro Arg Leu Cys Ala Gly     450 455 460 Ser Glu Leu Ala Lys Leu Glu Met Ala Val Phe Ile His His Leu Val 465 470 475 480 Leu Lys Phe Asn Trp Glu Leu Ala Glu Asp Asp Lys Pro Phe Ala Phe                 485 490 495 Pro Phe Val Asp Phe Pro Asn Gly Leu Pro Ile Arg Val Ser Arg Ile             500 505 510 Leu     <210> 6 <211> 361 <212> DNA <213> Arabidopsis thaliana <220> <221> misc_feature (222) (1) .. (361) <223> DWF4 fragment for RNAi <400> 6 gccggacatg agacttcttc tgtagccatt gctctcgcta tcttcttctt gcaagcttgc 60 cctaaagccg ttgaagagct tagggaagag catcttgaga tcgcgagggc caagaaggaa 120 ctaggagagt cagaattaaa ttgggatgat tacaagaaaa tggactttac tcaatgtgtt 180 ataaatgaaa ctcttcgatt gggaaatgta gttaggtttt tgcatcgcaa agcactcaaa 240 gatgttcggt acaaaggata cgatatccct agtgggtgga aagtgttacc ggtgatctca 300 gccgtacatt tggataattc tcgttatgac caacctaatc tctttaatcc ttggagatgg 360 c 361 <210> 7 <211> 1086 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS (222) (1) .. (1083) <223> SMT2 (GenBank accession no.NM_101884) <400> 7 atg gac tct tta aca ctc ttc ttc acc ggt gca ctc gtc gcc gtc ggt 48 Met Asp Ser Leu Thr Leu Phe Phe Thr Gly Ala Leu Val Ala Val Gly   1 5 10 15 atc tac tgg ttc ctc tgc gtt ctc ggt cca gca gag cgt aaa ggc aaa 96 Ile Tyr Trp Phe Leu Cys Val Leu Gly Pro Ala Glu Arg Lys Gly Lys              20 25 30 cga gcc gta gat ctc tct ggt ggc tca atc tcc gcc gag aaa gtc caa 144 Arg Ala Val Asp Leu Ser Gly Gly Ser Ile Ser Ala Glu Lys Val Gln          35 40 45 gac aac tac aaa cag tac tgg tct ttc ttc cgc cgt cca aaa gaa atc 192 Asp Asn Tyr Lys Gln Tyr Trp Ser Phe Phe Arg Arg Pro Lys Glu Ile      50 55 60 gaa acc gcc gag aaa gtt cca gac ttc gtc gac aca ttc tac aat ctc 240 Glu Thr Ala Glu Lys Val Pro Asp Phe Val Asp Thr Phe Tyr Asn Leu  65 70 75 80 gtc acc gac ata tac gag tgg gga tgg gga caa tcc ttc cac ttc tca 288 Val Thr Asp Ile Tyr Glu Trp Gly Trp Gly Gln Ser Phe His Phe Ser                  85 90 95 cca tca atc ccc gga aaa tct cac aaa gac gcc acg cgc ctc cac gaa 336 Pro Ser Ile Pro Gly Lys Ser His Lys Asp Ala Thr Arg Leu His Glu             100 105 110 gag atg gcc gta gat ctg atc caa gtc aaa cct ggt caa aag atc cta 384 Glu Met Ala Val Asp Leu Ile Gln Val Lys Pro Gly Gln Lys Ile Leu         115 120 125 gac gtc gga tgc ggt gtc ggc ggt ccg atg cga gcg att gca tct cac 432 Asp Val Gly Cys Gly Val Gly Gly Pro Met Arg Ala Ile Ala Ser His     130 135 140 tcg cga gct aac gta gtc ggg att aca ata aac gag tat cag gtg aac 480 Ser Arg Ala Asn Val Val Gly Ile Thr Ile Asn Glu Tyr Gln Val Asn 145 150 155 160 aga gct cgt ctc cac aat aag aaa gct ggt ctc gac gcg ctt tgc gag 528 Arg Ala Arg Leu His Asn Lys Lys Ala Gly Leu Asp Ala Leu Cys Glu                 165 170 175 gtc gtg tgt ggt aac ttc ctc cag atg ccg ttc gat gac aac agt ttc 576 Val Val Cys Gly Asn Phe Leu Gln Met Pro Phe Asp Asp Asn Ser Phe             180 185 190 gac ggt gct tat tcc atc gaa gcc acg tgt cac gcg ccg aag ctg gaa 624 Asp Gly Ala Tyr Ser Ile Glu Ala Thr Cys His Ala Pro Lys Leu Glu         195 200 205 gaa gtg tac gca gag atc tac agg gtg ttg aaa ccc gga tct atg tat 672 Glu Val Tyr Ala Glu Ile Tyr Arg Val Leu Lys Pro Gly Ser Met Tyr     210 215 220 gtg tcg tac gag tgg gtt acg acg gag aaa ttt aag gcg gag gat gac 720 Val Ser Tyr Glu Trp Val Thr Thr Glu Lys Phe Lys Ala Glu Asp Asp 225 230 235 240 gaa cac gtg gag gta atc caa ggg att gag aga ggc gat gcg tta cca 768 Glu His Val Glu Val Ile Gln Gly Ile Glu Arg Gly Asp Ala Leu Pro                 245 250 255 ggg ctt agg gct tac gtg gat ata gct gag acg gct aaa aag gtt ggg 816 Gly Leu Arg Ala Tyr Val Asp Ile Ala Glu Thr Ala Lys Lys Val Gly             260 265 270 ttt gag ata gtg aag gag aag gat ctg gcg agt cca ccg gct gag ccg 864 Phe Glu Ile Val Lys Glu Lys Asp Leu Ala Ser Pro Pro Ala Glu Pro         275 280 285 tgg tgg act agg ctt aag atg ggt agg ctt gct tat tgg agg aat cac 912 Trp Trp Thr Arg Leu Lys Met Gly Arg Leu Ala Tyr Trp Arg Asn His     290 295 300 att gtg gtt cag att ttg tca gcg gtt gga gtt gct cct aaa gga act 960 Ile Val Val Gln Ile Leu Ser Ala Val Gly Val Ala Pro Lys Gly Thr 305 310 315 320 gtt gat gtt cat gag atg ttg ttt aag act gct gat tat ttg acc aga 1008 Val Asp Val His Glu Met Leu Phe Lys Thr Ala Asp Tyr Leu Thr Arg                 325 330 335 gga ggt gaa acc gga ata ttc tct ccg atg cat atg att ctc tgc aga 1056 Gly Gly Glu Thr Gly Ile Phe Ser Pro Met His Met Ile Leu Cys Arg             340 345 350 aaa ccg gag tca ccg gag gag agt tct tga 1086 Lys Pro Glu Ser Pro Glu Glu Ser Ser         355 360 <210> 8 <211> 361 <212> PRT <213> Arabidopsis thaliana <400> 8 Met Asp Ser Leu Thr Leu Phe Phe Thr Gly Ala Leu Val Ala Val Gly   1 5 10 15 Ile Tyr Trp Phe Leu Cys Val Leu Gly Pro Ala Glu Arg Lys Gly Lys              20 25 30 Arg Ala Val Asp Leu Ser Gly Gly Ser Ile Ser Ala Glu Lys Val Gln          35 40 45 Asp Asn Tyr Lys Gln Tyr Trp Ser Phe Phe Arg Arg Pro Lys Glu Ile      50 55 60 Glu Thr Ala Glu Lys Val Pro Asp Phe Val Asp Thr Phe Tyr Asn Leu  65 70 75 80 Val Thr Asp Ile Tyr Glu Trp Gly Trp Gly Gln Ser Phe His Phe Ser                  85 90 95 Pro Ser Ile Pro Gly Lys Ser His Lys Asp Ala Thr Arg Leu His Glu             100 105 110 Glu Met Ala Val Asp Leu Ile Gln Val Lys Pro Gly Gln Lys Ile Leu         115 120 125 Asp Val Gly Cys Gly Val Gly Gly Pro Met Arg Ala Ile Ala Ser His     130 135 140 Ser Arg Ala Asn Val Val Gly Ile Thr Ile Asn Glu Tyr Gln Val Asn 145 150 155 160 Arg Ala Arg Leu His Asn Lys Lys Ala Gly Leu Asp Ala Leu Cys Glu                 165 170 175 Val Val Cys Gly Asn Phe Leu Gln Met Pro Phe Asp Asp Asn Ser Phe             180 185 190 Asp Gly Ala Tyr Ser Ile Glu Ala Thr Cys His Ala Pro Lys Leu Glu         195 200 205 Glu Val Tyr Ala Glu Ile Tyr Arg Val Leu Lys Pro Gly Ser Met Tyr     210 215 220 Val Ser Tyr Glu Trp Val Thr Thr Glu Lys Phe Lys Ala Glu Asp Asp 225 230 235 240 Glu His Val Glu Val Ile Gln Gly Ile Glu Arg Gly Asp Ala Leu Pro                 245 250 255 Gly Leu Arg Ala Tyr Val Asp Ile Ala Glu Thr Ala Lys Lys Val Gly             260 265 270 Phe Glu Ile Val Lys Glu Lys Asp Leu Ala Ser Pro Pro Ala Glu Pro         275 280 285 Trp Trp Thr Arg Leu Lys Met Gly Arg Leu Ala Tyr Trp Arg Asn His     290 295 300 Ile Val Val Gln Ile Leu Ser Ala Val Gly Val Ala Pro Lys Gly Thr 305 310 315 320 Val Asp Val His Glu Met Leu Phe Lys Thr Ala Asp Tyr Leu Thr Arg                 325 330 335 Gly Gly Glu Thr Gly Ile Phe Ser Pro Met His Met Ile Leu Cys Arg             340 345 350 Lys Pro Glu Ser Pro Glu Glu Ser Ser         355 360 <210> 9 <211> 288 <212> DNA <213> Arabidopsis thaliana <220> <221> misc_feature (222) (1) .. (288) <223> SMT2 fragment for RNAi <400> 9 ctagacgtcg gatgcggtgt cggcggtccg atgcgagcga ttgcatctca ctcgcgagct 60 aacgtagtcg ggattacaat aaacgagtat caggtgaaca gagctcgtct ccacaataag 120 aaagctggtc tcgacgcgct ttgcgaggtc gtgtgtggta acttcctcca gatgccgttc 180 gatgacaaca gtttcgacgg tgcttattcc atcgaagcca cgtgtcacgc gccgaagctg 240 gaagaagtgt acgcagagat ctacagggtg ttgaaacccg gatctatg 288 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DET2 sense primer <400> 10 atggaagaaa tcgccgataa aac 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DET2 anti-sense primer <400> 11 tcagtacaca aaaggaataa cag 23 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DWF4 sense primer <400> 12 atgttcgaaa cagagcatc 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DWF4 anti-sense primer <400> 13 ttacagaata cgagaaaccc 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SMT2 sense primer <400> 14 atggactctt taacactctt c 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SMT2 anti-sense primer <400> 15 tcaagaactc tcctccggtg 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for DET2 RNAi <400> 16 aagctttcat ttaccctctt cg 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for DET2 RNAi <400> 17 gtcgacgaac ttggcaatgt ac 22 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer for DWF4 RNAi <400> 18 gtcgacgccg gacatgagac ttc 23 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for DWF4 RNAi <400> 19 ggtaccgcca tctccaagga ttaa 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer for SMT2 RNAi <400> 20 ggtaccctag acgtcggatg cggt 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for SMT2 RNAi <400> 21 gaattccata gatccgggtt tcaa 24 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> sense primer for DET2: DWF4: SMT2 <400> 22 tctagaggcg cgcctcattt accctcttcg c 31 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for DET2: DWF4: SMT2 <400> 23 ggatccattt aaatcataga tccgggtttc aa 32 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T3 qRT-PCR (DET2) <400> 24 atggaagaaa tcgccgataa aacc 24 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for T3 qRT-PCR (DET2) <400> 25 acacaaaagg aataacagct ttac 24 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T3 qRT-PCR (DWF4) <400> 26 atgttcgaaa cagagcatca tac 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for T3 qRT-PCR (DWF4) <400> 27 gaatacgaga aaccctaata ggc 23 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T3 qRT-PCR (SMT2) <400> 28 atggactctt taacactctt c 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for T3 qRT-PCR (SMT2) <400> 29 agaactctcc tccggtgact c 21 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T3 qRT-PCR (CYP85A1) <400> 30 gctcgcttct gtctctca 18 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for T3 qRT-PCR (CYP85A1) <400> 31 tcatcccctc ctatttcc 18 <210> 32 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> sense primer for 18s rRNA <400> 32 ggatgggtcg gccggtc 17 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> anti sense primer for 18s rRNA <400> 33 caggctgagg tctcgttc 18 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T4 qRT-PCR (DET2) <400> 34 tttggagagg cgattgagtg 20 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> anti-sense primer for T4 qRT-PCR (DET2) <400> 35 ctcttccttg aacttggcaa tg 22 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T4 qRT-PCR (DWF4) <400> 36 cagaggatga agcagagatg ag 22 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti sense primer for T4 qRT-PCR (DWF4) <400> 37 tgagatcgag aatttgctcc g 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T4 qRT-PCR (SMT2) <400> 38 ccaaaagaaa tcgaaaccgc c 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> anti sense primer for T4 qRT-PCR (SMT2) <400> 39 gcgtctttgt gagattttcc g 21 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for T4 qRT-PCR (CYP85A1) <400> 40 acagagcaga aaacagagtg ag 22 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> anti sense primer for qRT-PCR (CYP85A1) <400> 41 gaaggagagc ggaacagag 19

Claims (10)

A herbicide resistant selection marker, a promoter, a spacer and a terminator between two T-DNA borders,
Sense bases for RNA interference of De-Etilolated 2 (det2) genes linked to each other, sense bases for RNA interference of dwarf 4 (dwf4) gene, and sense bases for RNA interference of sterol methyltransferase 2 (smt2) gene The sequences and their respective antisense sequences linked adjacent to each other are arranged in reverse order with spacers interposed therebetween, and operably linked with a promoter,
RNA interference recombinant vector for plant expression. According to claim 1, wherein the sense base for RNA interference of the De-Etilolated 2 (det2) gene is DNA of the nucleotide sequence of SEQ ID NO: 3, the sense base sequence for RNA interference of the dwarf 4 (dwf4) gene is SEQ ID NO: DNA of the nucleotide sequence of 6, the sense sequence for RNA interference of the sterol methyltransferase 2 (smt2) gene is DNA of the nucleotide sequence of SEQ ID NO: 9, each of these antisense sequences are complementary to each of the sense sequences RNA interference recombinant vector for plant expression which is DNA of sequence. The RNA interference recombinant vector for plant expression according to claim 2, wherein the RNA interference recombinant vector is a pFGC5941- De-Etilolated 2 (det2): dwarf 4 (dwf4): sterol methyltransferase 2 (smt2) vector described in FIG. 1A. A microorganism transformed with the vector according to any one of claims 1 to 3. The transformed microorganism of claim 4, wherein the microorganism is Agrobacterium tumefaciens. Arabidopsis transformed with the vector of any one of claims 1 to 3. 7. The transformed Arabidopsis larvae of claim 6, wherein the Arabidopsis oleracea contains an increased amount of camphorsterol compared to the wild type. Method for producing camphorsterol, characterized in that extracted from the transformed Arabidopsis of claim 6. A method for increasing camphorsterol content in Arabidopsis, comprising transforming with a vector according to any one of claims 1 to 3. A Arabidopsis extract containing camphorsterol, which is extracted from the transformed Arabidopsis recited in claim 6.

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