KR20150109321A - RcLPAT376 genes specific to Ricinus communis L. anther and their uses in hydroxy fatty acid - Google Patents

Abstract

The invention castor (Ricinus communis L.) and the production of 12-hydroxy-octadeca-9-enoic acid by the use of the RcLPAT376 gene, The content of the hydroxy fatty acid in the seeds of the transgenic plant transformed with the RcLPAT376 gene is improved, so that the gene can be usefully used for increasing the production of hydroxy fatty acid in various plants.

Description

RcLPAT376 genes specific to Ricinus communis L. anther and their uses in hydroxy fatty acid {RcLPAT376 genes specific to Ricinus communis L. anther and their uses in castor surgery}

The present invention relates to a castor surgery-specific RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene and its use in the production of hydroxy fatty acids, and more particularly, to a RcLPAT376 gene that is surgically-specific and functions as an LPAT (Lysophosphatidyl acyltransferase). It was isolated from castor and relates to the effect of increasing the hydroxy fatty acid content through the expression of the RcLPAT376 gene.

The main component of vegetable oil is a fatty acid having 16 or 18 carbon chains, and is a mixture of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid, It is suitable for use in frying or in salads, but it has the disadvantage that it is not suitable for industrial use. Currently, 80% of the world's vegetable oils are consumed for food and the remaining 20% for industrial use. In order for vegetable oils to be economical for industrial use in the future, materials must be suitable and have good processability. For example, in order to be used as a lubricant, oxidation must be difficult, the viscosity must be high, and in the case of paints or paints, it must be easily oxidized.

On the other hand, seeds of uncultivated plant species contain hundreds of different fatty acids, and these fatty acids are called unusual fatty acids, distinguishing them from five types of fatty acids, which are the main constituents of general vegetable oils. These specific fatty acids have specific properties due to a suitable number of carbon chains suitable for industrial use, a suitable degree of unsaturation, and a functional group having various activities on carbon (e.g., a hydroxy group (-OH), an epoxy group). In particular, the delta 12 of castor Hydroxy fatty acids (ricinoleic acid; ricinoleic acid) account for 90% of seed fatty acids, and processed by processing them are used in a wide range of commercial and industrial products, including high-quality lubricants, nylons, and cosmetics.

However, non-cultivated plants that produce specific fatty acids such as castor have the disadvantages that the yield is small, flowering is uneven, and it is difficult to cultivate industrially due to the toxin called ricin in the seeds. Bioengineering research and techniques to separate molecules and introduce them into crops are being concentrated, but the efficiency of producing specific fatty acids is very low in transgenic plants.

Accordingly, the present inventors searched for a gene that increases hydroxy fatty acid from castor, isolated castor RcLPAT376 gene specifically expressed in male flowers, and confirmed the effect of increasing hydroxy fatty acid content through expression of the RcLPAT376 gene, the present invention Was completed.

An object of the present invention is to provide a recombinant vector containing the RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene used for the production of hydroxy fatty acid consisting of the nucleotide sequence of SEQ ID NO: 1.

In addition, another object of the present invention is to provide a transgenic plant transformed with the recombinant vector.

In addition, another object of the present invention is to provide a method for producing the transgenic plant.

In addition, another object of the present invention is to provide a method for producing hydroxy fatty acids.

In order to achieve the above object, the present invention provides a recombinant vector comprising the RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene used for the production of hydroxy fatty acid consisting of the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides a transgenic plant transformed with the recombinant vector.

In addition, the present invention

(1) separating the RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene from castor (Ricinus communis L.) using the primer set represented by SEQ ID NOs: 3 and 4;

(2) preparing a recombinant vector including the RcLPAT376 gene consisting of the nucleotide sequence of SEQ ID NO: 1 isolated in step (1);

(3) transforming the vector into a plant using Agrobacterium;

(4) selecting a transformed plant by spraying a BASTA herbicide;

(5) obtaining T1 seeds from the selected transgenic plants;

(6) selecting a line having an increased content of hydroxy fatty acid in the T1 seeds of step (5);

(7) developing the line selected in step (6) to the T2 generation;

(8) selecting a line having an increased content of hydroxy fatty acid in the T2 seeds of step (7); And

(9) A method for producing a hydroxy fatty acid comprising the step of obtaining a hydroxy fatty acid from the seed of the T3 generation of step (8),

The hydroxy fatty acid provides a method for producing a hydroxy fatty acid, characterized in that the hydroxy fatty acid is 12-hydroxy-octadeca-9-enoic acid.

Since the content of hydroxy fatty acids in seeds of plants transformed with the vector containing the castor surgery-specific RcLPAT376 gene of the present invention has been improved, the gene can be used to increase the production of hydroxy fatty acids in various plants. .

1 is a diagram showing the result of comparing the amino acid sequence of the protein encoded by the isolated castor RcLPAT376 gene and the Arabidopsis AtLPAT2 protein, which was previously found to have an LPAT function;
RcLPAT376 shows a low 48.7% homology with Arabidopsis AtLPAT2, and the homology part is shown in gray.
Figure 2 is a diagram showing the expression of the castor ACTIN2 (RcACT2) gene as a control, the expression RT-PCR analysis of the castor RcLPAT376 gene for each castor tissue;
L (leaf), St (stem), UF (whole flower), FF (female flower), MF (male flower), S (seed), Sd (seedling) and R (root) tissue-specific expressions are shown.
Figure 3a is a schematic diagram showing a recombinant vector for E. coli transformation containing the RcLPAT376 gene and GUS gene;
T7P is the T7 promoter, 6His is bound to the N-terminal of RcLPAT376 and GUS, T7T is the T7 terminator, Amp ^R is the ampicillin resistance gene.
3B is a graph showing the results of comparative analysis of the growth of the LPAT mutant JC201 E. coli in which the vector was transformed with pDEST-RcLPAT376 and pDEST-GUS.
Figure 3c is a diagram showing the result of detecting the RcLPAT376 protein in JC201 E. coli.
Fig. 4 is a diagram showing the structure of a plant vector expressing a specific expression of a castor phaseolin-RcLPAT376 seed;
LB: left border, RB: right border, bar: herbicide phosphitotricin resistance gene, T35S: terminator of CaMV 35 gene and SpR: spectinomycin Resistance gene.
5A is a graph showing the percentage of hydroxy fatty acids in T2 seeds transformed with RcLPAT376 gene.
5B is a graph showing the percentage of hydroxy fatty acids in T3 seeds transformed with the RcLPAT376 gene.
6 is a diagram showing the results of TLC (Thin-Layer Chromatography) analysis of seed fat in the Arabidopsis thaliana transformed with CL37 Arabidopsis (control) and CL37+RcLPAT376 producing hydroxy fatty acids.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant vector containing the RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene used for the production of hydroxy fatty acid consisting of the nucleotide sequence of SEQ ID NO: 1.

The gene is castor ( Ricinus communis It is preferable to be separated from L.). It is not limited to this.

The gene is preferably expressed in male flowers, but is not limited thereto.

The gene preferably encodes a protein consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In addition, variants of the base sequence are included within the scope of the present invention. Specifically, the gene is a base sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 1 Can include. The "% of sequence homology" for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

The hydroxy fatty acid is 12-hydroxy-octadeca-9-enophosphoric acid, characterized by a structure including one double bond on carbon 9 and one hydroxyl group on carbon 12 on a fatty acid chain having 18 carbon atoms. (12-hydroxy-octadeca-9-enoic acid) is preferred, but is not limited thereto.

The recombinant vector is preferably for promoting hydroxy fatty acid, but is not limited thereto.

The recombinant vector preferably contains the herbicide resistance Bar gene and the RcLPAT376 gene is located under the seed-specific Phaseoline promoter, but is not limited thereto.

In addition, the present invention provides a recombinant vector comprising the RcLPAT376 gene according to the present invention. The recombinant vector may preferably be the Phaseoline-RcLPAT376 vector described in FIG. 4, but is not limited thereto.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, recombinant cells can express genes found in cells in their natural state, but the genes are modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment(s), a nucleic acid molecule, which is delivered into a cell. Vectors replicate DNA and can be reproduced independently in host cells. The term “expression vector” is often used interchangeably with “recombinant vector”. The term “recombinant vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals usable in eukaryotic cells are known.

The vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the recombinant vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pLλ promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , It is common to include a ribosome binding site for initiation of translation and a transcription/translation termination sequence.

On the other hand, vectors that can be used in the present invention include plasmids often used in the art (e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (e.g. , λ-Charon, λΔz1 and M13, etc.) or a virus (eg, SV40, etc.).

The vector of the present invention may contain an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin. , Tetracycline and BASTA herbicide resistance genes.

In the present invention, a plant transformed with a castor surgery-specific RcLPAT376 gene having resistance to BASTA was selected.

In addition, the present invention provides a transgenic plant transformed with the recombinant vector.

In the plant, the content of hydroxy fatty acids in seeds is preferably increased by expression of the RcLPAT376 gene, T1 seeds are more preferable, T2 seeds are more preferable, and T3 seeds are most preferable, but the present invention is not limited thereto.

The plant is preferably Arabidopsis or a vegetable oil plant, but is not limited thereto.

The oil plant is preferably a plant that obtains oil from seeds or fruits, and is more preferably selected from the group consisting of sesame seeds, rapeseed, sunflowers, soda, peanuts, beans, coco palms, oil palms, and jojoba, but is not limited thereto. .

In the recombinant vector of the present invention, the promoter may be a CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but is not limited thereto. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation. Since the selection of transformants can be made by various tissues at various stages, constitutive promoters may be preferred in the present invention. Thus, constitutive promoters do not limit the possibility of selection.

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

Plant transformation refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have periods of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both monocotyledons as well as dicotyledons. In principle, any transformation method can be used to introduce hybrid DNA according to the present invention into suitable progenitor cells. The method is the 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), protoplasts. Of electroporation (Shillito RD et al., 1985 Bio/Technol. 3, 1099-1102), microinjection with plant urea (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) ), (DNA or RNA-coated) particle bombardment method of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumerfasi by transformation of plant infiltration or mature pollen or vesicle In Ens-mediated gene transfer, it can be appropriately selected from (incomplete) virus-induced infection (EP 0 301 316) and the like. A preferred method according to the invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector technology as described in EP A 120 516 and U.S. Patent No. 4,940,838.

The plant according to the present invention is a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; Specialty crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots, and bananas; Flowers selected from the group consisting of roses, gladiolus, gerbera, carnations, chrysanthemums, lilies and tulips; And it may be a feed crop selected from the group consisting of ryegrass, red clover, orchardgrass, alpha alpha, tolpescue, and perennial ryegrass.

In addition, the present invention

(1) separating the RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene from castor (Ricinus communis L.) using the primer set represented by SEQ ID NOs: 3 and 4;

(2) preparing a recombinant vector including the RcLPAT376 gene consisting of the nucleotide sequence of SEQ ID NO: 1 isolated in step (1);

(3) transforming the vector into a plant using Agrobacterium;

(4) selecting a transformed plant by spraying a BASTA herbicide;

(5) obtaining T1 seeds from the selected transgenic plants;

(6) selecting a line having an increased content of hydroxy fatty acid in the T1 seeds of step (5);

(7) developing the line selected in step (6) to the T2 generation;

(8) selecting a line having an increased content of hydroxy fatty acid in the T2 seeds of step (7); And

(9) A method for producing a hydroxy fatty acid comprising the step of obtaining a hydroxy fatty acid from the seed of the T3 generation of step (8),

The hydroxy fatty acid provides a method for producing a hydroxy fatty acid, characterized in that the hydroxy fatty acid is 12-hydroxy-octadeca-9-enoic acid.

In a specific embodiment of the present invention, the present inventors isolated the RcLPAT376 gene, which is surgically expressed and functions as LPAT (Lysophosphatidyl acyltransferase), from castors, and the RcLPAT376 of castors was combined with a promoter of the seed-specific gene, Phaseolin, and hides from the seeds. It was confirmed that the hydroxy fatty acid was enhanced up to 22.3% by transforming CL37 Arabidopsis plants that produce up to 17% of hydroxy fatty acids (Table 2, FIGS. 5 and 6).

Therefore, since the plant transformed with the vector containing the castor surgery-specific RcLPAT376 gene of the present invention improved generation, the content of hydroxy fatty acid in the seed was significantly improved, and thus the hydroxy fatty acid in various plants using the gene It can be usefully used to increase the production of.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

<Example 1> LPAT376 gene isolation from castor

The present inventors, castor ( Ricinus communis After synthesizing complementary DNA from RNA isolated from L.), the LPAT376 gene was isolated using a primer sequence specific to the RcLPAT376 gene.

Specifically, complementary DNA for all RNAs expressed in leaves and seeds was synthesized from RNA isolated from leaves and seeds of castor using PrimeScript ^{TM 1st strand cDNA Synthesis Kit (TakaRa, Japan).} Primers were prepared as shown in Table 1 below, and PCR (polymerase chain reaction) was performed using KOD+ polymerase (Toyobo, Japan) using this to synthesize RcLPAT376 cDNA. The synthesized RcLPAT376 cDNA was cloned into a pENTR/D-Topo (Invitrogen, Carlsbad, CA, USA) vector to prepare a pENTR-LPAT376 vector. The nucleic acid sequence of the cloned RcLPAT376 was determined.

As a result, the nucleic acid sequence of the RcLPAT376 cDNA gene was composed of a total of 1,131 bp (SEQ ID NO: 1), and was composed of 43 kDa molecular weight protein information composed of 376 amino acids (SEQ ID NO: 2). As a result of a homology analysis between the protein sequence of the RcLPAT376 gene and Arabidopsis thaliana LPAT2 (Kim et al., Plant Cell 17: 1073-1089, 2005) whose function of LPAT (Lysophosphatidyl acyltransferase) was confirmed, a 48.7% lower homology was performed. Shows that the LPAT enzyme function could not be deduced from the protein sequence alone (FIG. 1 ). The isolated RcLPAT376 gene was identical to the protein sequence of XP_002513486 registered in NCBI as a result of protein homology by BlastP of NCBI (National Center for Biotechnology Information). This protein was presumed to be 1-acylglycerol-3-phosphate acyltransferase, but its function was not reported at all.

Sequence number Sequence name Sequence (5'->3') 3 RcLPAT376 forward primer 5'-CACCATGGAAGTTAAGGCTGTTCTTC-3' 4 RcLPAT376 reverse primer 5'-TTAGTTTTTCAGTTGCACTGTTAAATTTCC-3'

< Example 2> Separated from casters RcLPAT376 Gene expression analysis

The present inventors analyzed the expression of the RcLPAT376 gene isolated from castor by tissue in castor by RT-PCR (Reverse transcriptase-polymerase chain reaction) method.

Specifically, RNA from leaves, stems, flowers, female flowers, male flowers, seeds, seedlings, and roots of castor was isolated, and then RcLPAT376 gene-specific primers were prepared and used for RT-PCR to analyze the expression pattern of each tissue. It is shown in Table 1 above.

As a result, the RcLPAT376 gene was strongly expressed in flowers, especially strongly expressed in male flowers, and was very weak or not expressed at all in other tissues (FIG. 2). In order to prove accurate expression analysis, the RcACT2 gene expression analysis of the actin gene expressed in all tissues of castor was used as a control.

<Example 3> Analysis of enzyme function of LPAT376 gene isolated from castor

<3-1> Preparation of RcLPAT376 gene expression vector

The present inventors prepared a castor RcLPAT376 gene expression vector to test whether LPAT376 isolated from castor functions as an LPAT enzyme.

Specifically, pDEST-RcLPAT376 was produced by cloning RcLPAT376 into E. coli expression vector pDEST17 (Invitrogen, Carlsbad, CA, USA) containing a T7 promoter so that the castor RcLPAT376 gene can be expressed in E. coli. In addition, as a control for the E. coli expression experiment, the GUS gene was cloned into the same pDEST vector to produce a pDEST-GUS expression vector (FIG. 3A).

<3-2> RcLPAT376 gene transformation and enzyme function analysis

The present inventors transformed the plasmid containing the RcLPAT376 gene to confirm that castor RcLPAT376 has the function of the LPAT enzyme.

Specifically, the pDEST-RcLPAT376 plasmid containing the RcLPAT376 gene was transformed into E. coli JC201 to determine whether castor RcLPAT376 has the function of the LPAT enzyme. In addition, E. coli JC201 was transformed using GUS as a control gene. E. coli JC201 into which the control gene GUS and the RcLPAT376 gene were introduced were selected and cultured at 30°C and 42°C, and their growth was compared at an absorbance of 600 nm (OD 600) over time.

As a result, JC201 E. coli into which the GUS gene was introduced normally grew well up to an OD of 0.4 at 30°C, but showed a value of less than 0.05 OD at 42°C and failed to grow. However, JC201, into which the castor RcLPAT376 gene was introduced, exhibited an OD of 0.6 or higher at 42° C., which is the lethal temperature of JC201 E. coli, as well as 30° C. and grew normally (FIG. 3b ). At 42°C, which is a high temperature, JC201 is killed due to the inactivation of LPAT and cannot grow. When the GUS gene was introduced into JC201, E. coli could not grow because the function of LPAT was not as expected. On the contrary, in JC201 to which RcLPAT376 was introduced, RcLPAT376 functioned as LPAT to induce normal growth of JC201 E. coli even at 42° C. high temperature (Fig. 3b).

<3-3> Analysis of castor RcLPAT376 protein derived

The present inventors detected the RcLPAT376 protein to confirm that the growth of JC201 E. coli was not killed at 42° C. and recovered by the introduction of RcLPAT376 was derived by expression of the RcLPAT376 protein.

Specifically, all proteins were isolated from JC201 E. coli and subjected to protein electrophoresis. After the electrophoresed protein was transferred to a PVDF membrane, the RcLPAT376 protein bound to His-tag was analyzed by Western blot using a His-tag antibody.

As a result, it was confirmed that the 42.7 kDa His-RcLPAT375 protein was expressed in the transformed JC201 E. coli (FIG. 3C).

<Example 4> RcLPAT376 seed-specific expression plant vector construction

In order to construct a castor RcLPAT376 seed-specific expression plant vector, a plant recombinant expression vector expressing the RcLPAT376 gene was constructed under the control of a seed-specific phaseolin promoter.

Specifically, the pENTR-RcLPAT376 vector into which the castor RcLPAT376 gene was inserted and the plant expression vector pB2GW7 (10.8 kb) purchased from VIB-Ghent University (Karimi et al., Trend Plant Sci. 7: 193-195, 2002). Recombinant expression of plants in which the RcLPAT376 gene is expressed under the control of the paseolin promoter by replacing the promoter with the seed-specific paseolin promoter (Chandrasekharan et al. Plant J. 33:853-866, 2002) and reacting with pPhaseolin-gateway created by LR clonase reaction. The vector Phaseolin-RcLPAT376 was constructed (Fig. 4).

The plant expression vector prepared above was transformed into Agrobacterium GV3101 (Komcz and Schell. Mol. Gen. Genet. 204:383-396. 1986), and Agrobacterium was used for transformation of Arabidopsis plants (Komcz and Schell. Mol. Gen. Genet. 204:383-396. Clough and Bent, Plant J. 16:735-743, 1998).

< Example 5> derived from castor RcLPAT376 By gene expression Hydroxy Fatty Acid Enhancement Analysis

In order to analyze the enhancement of hydroxy fatty acid by the specific expression of the castor RcLPAT376 gene, hydroxy in Arabidopsis thaliana transformed with the castor RcLPAT376 gene. Fatty acid was analyzed.

Specifically, CL37 Arabidopsis (Lu et al., Plant J. 45:847-856, 2006; Kumar et al., Plant J. 48:920-930, 2007). After the next generation seeds were harvested and germinated, T1 Arabidopsis thaliana transformed with the RcLPAT376 gene of castor resistant to herbicide (BASTA) was selected. The fatty acid content of T2 seeds obtained from T1 transformant plants was analyzed by Gas Chromotography (GC) to investigate the content of hydroxy fatty acid. For the CL37 Arabidopsis control and the plants transformed with LPAT376 in CL37, T2 seeds were harvested from 10 independent T1 plants and analyzed for fatty acids. In addition, the average of the hydroxy fatty acid production of the three T3 seeds was analyzed in triplicate (n=3) to see if the increase in hydroxy fatty acid production of the T2 transformants was maintained in the next generation by RcLPAT376.

As a result, the hydroxy fatty acid of CL37 Arabidopsis seed showed 17% production as previously reported, but RcLPAT376 in CL37 showed higher productivity than CL37 hydroxy fatty acid in transformant seeds, on average 19.6%. By looking at, the production of hydroxy fatty acids was at least 18.2% and up to 21.7%. This result shows that the RcLPAT376 gene has an effect of enhancing the production of hydroxy fatty acids (FIG. 5A).

In addition, after harvesting T3 seeds from the T2 plants of the transformants #19 and #31, which had the most hydroxy fatty acids, the fatty acids of the seeds were analyzed, and the production of hydroxy fatty acids was 21.4% to 22.3%. This result confirmed that the production enhancing effect of up to 34% was induced by the introduction of the RcLPAT376 gene, based on the production of 16.6% of hydroxy fatty acid in the CL37 control (Table 2 and FIG. 5B).

< Example 6> To LPAT376 Of seeds by Hydroxy Analysis of the relationship between fatty acid production and seed fat production

To investigate whether the increase in hydroxy fatty acid production of seeds by LPAT376 was related to the increase in fat production of seeds, fat was isolated from control CL37 and transformant CL37+LPAT376 seeds and then analyzed by TLC (Thin-Layer Chromatography).

Specifically, fat was isolated from 30 T2 seeds obtained from line #19 of CL37 control and transformant CL37+RcLPAT376 and analyzed by TLC (Thin-Layer Chromatography).

As a result, lipids were separated from the T2 seed obtained from the #19 line of CL37 and the transformant CL37+RcLPAT376 and developed by TLC. TAG increased by about 2 times or more than the control, CL37 (FIG. 6). These results show that LPAT376 efficiently accumulated hydroxy fatty acids in TAG.

Control CL37 and CL37 LPAT376 Fatty acid analysis of gene-transformed T3 seeds n=3 Average of fatty acid composition (mol%) n=3 Sum of HFAs
(18:1-OH+18:2-OH) HFAs
Rate of increase (%) 16:0 18:0 18:1 18:2 18:3 20:0 20:1 18:1-OH 18:2
-OH CL37 14.6 7.2 34.3 20.4 5.5 0.9 0.4 13.4 3.2 16.6 0.0 CL37+RcLPAT376 (T3 of 19-1) 13.5 6.6 27.5 22.3 6.6 1.0 0.4 17.4 4.6 22.0 32.5 CL37+RcLPAT376 (T3 of 19-2) 13.9 6.9 27.7 22.0 6.8 1.0 0.5 17.2 4.2 21.4 28.9 CL37+RcLPAT376 (T3 of 31-2) 14.1 6.7 27.3 21.5 6.7 1.0 0.4 18.1 4.2 22.3 34.3

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> RcLPAT376 genes specific to Ricinus communis L. anther and their uses in hydroxy fatty acid <130> P15R12D1266 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 1131 <212> DNA <213> RcLPAT376 cDNA <400> 1 atggaagtta aggctgttct tctctctatt ccttttggct ttgtttttct tttcagtggc 60 cttatcatca atctaatcca aggagcttgc tacatcctag tacagcctct atctaaaact 120 gcacacagaa ggatcattgg agcggtgacg gaaatcttat ggctagagtt catatttctc 180 atggactggt ggtcaggctt taaggttcat ctccatacag attttgagac atatcaattg 240 atgggtaaag agcatgcact tctcatgcct aatcacatct gtgatgctga tatatttatt 300 atgtggcttc tggctcagcg ttcttcttgc ctccgtggcg cgctaatggt ggtgaagaga 360 tcttccatgt atattccgat ttatgggtgg gcaacttggt ttgctgagca tgtatttgta 420 agtagaaact ggggcaaaga tgaagaggtc ttgaagtcaa ggtttcaaag cctccaggat 480 ttcccaaggc ctttctggtt gacccttttt gtggaaggaa ctcgtatgac ctcagataaa 540 ctagtagcag ctcaaaattt cgccacttcc aagggtttgc cagttcctag gaatgtgttg 600 atccctcgta ccaaggggtt tgtggcagca gtacgacata tgcgttcatt tgttccagca 660 atttatgata ttacgctagc tgtaccgaga ggtcttcgaa ccccttctct gctaggattt 720 ttagggaggg agcctactgc tgtccaaatt cacataaagc gatactcgat gaaagagtta 780 ccagagtcag atgaagccgt tgctcaatgg tgcagagata agtttgttgc taaggataat 840 atgttggatg aatttcaagc gaaaggcaca tttgaaaacc aaaaaaatac agatattggt 900 cgaccaagaa agtcattgat tgtccttacc actttgggtt gcattatctg tctggtagta 960 atcagcttga ttcaaaggta ttccctgcta tctaccagga aaggcgtcat ctcattggca 1020 gctgggttgg cacttgatgc agtattcttg catgctgtaa ttgagtacac aaaattgcca 1080 caagccagaa aagcttccat gggaaattta acagtgcaac tgaaaaacta a 1131 <210> 2 <211> 376 <212> PRT <213> RcLPAT376 amino acid <400> 2 Met Glu Val Lys Ala Val Leu Leu Ser Ile Pro Phe Gly Phe Val Phe 1 5 10 15 Leu Phe Ser Gly Leu Ile Ile Asn Leu Ile Gln Gly Ala Cys Tyr Ile 20 25 30 Leu Val Gln Pro Leu Ser Lys Thr Ala His Arg Arg Ile Ile Gly Ala 35 40 45 Val Thr Glu Ile Leu Trp Leu Glu Phe Ile Phe Leu Met Asp Trp Trp 50 55 60 Ser Gly Phe Lys Val His Leu His Thr Asp Phe Glu Thr Tyr Gln Leu 65 70 75 80 Met Gly Lys Glu His Ala Leu Leu Met Pro Asn His Ile Cys Asp Ala 85 90 95 Asp Ile Phe Ile Met Trp Leu Leu Ala Gln Arg Ser Ser Cys Leu Arg 100 105 110 Gly Ala Leu Met Val Val Lys Arg Ser Ser Met Tyr Ile Pro Ile Tyr 115 120 125 Gly Trp Ala Thr Trp Phe Ala Glu His Val Phe Val Ser Arg Asn Trp 130 135 140 Gly Lys Asp Glu Glu Val Leu Lys Ser Arg Phe Gln Ser Leu Gln Asp 145 150 155 160 Phe Pro Arg Pro Phe Trp Leu Thr Leu Phe Val Glu Gly Thr Arg Met 165 170 175 Thr Ser Asp Lys Leu Val Ala Ala Gln Asn Phe Ala Thr Ser Lys Gly 180 185 190 Leu Pro Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val 195 200 205 Ala Ala Val Arg His Met Arg Ser Phe Val Pro Ala Ile Tyr Asp Ile 210 215 220 Thr Leu Ala Val Pro Arg Gly Leu Arg Thr Pro Ser Leu Leu Gly Phe 225 230 235 240 Leu Gly Arg Glu Pro Thr Ala Val Gln Ile His Ile Lys Arg Tyr Ser 245 250 255 Met Lys Glu Leu Pro Glu Ser Asp Glu Ala Val Ala Gln Trp Cys Arg 260 265 270 Asp Lys Phe Val Ala Lys Asp Asn Met Leu Asp Glu Phe Gln Ala Lys 275 280 285 Gly Thr Phe Glu Asn Gln Lys Asn Thr Asp Ile Gly Arg Pro Arg Lys 290 295 300 Ser Leu Ile Val Leu Thr Thr Leu Gly Cys Ile Ile Cys Leu Val Val 305 310 315 320 Ile Ser Leu Ile Gln Arg Tyr Ser Leu Leu Ser Thr Arg Lys Gly Val 325 330 335 Ile Ser Leu Ala Ala Gly Leu Ala Leu Asp Ala Val Phe Leu His Ala 340 345 350 Val Ile Glu Tyr Thr Lys Leu Pro Gln Ala Arg Lys Ala Ser Met Gly 355 360 365 Asn Leu Thr Val Gln Leu Lys Asn 370 375 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RcLPAT376 forward primer <400> 3 caccatggaa gttaaggctg ttcttc 26 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> RcLPAT376 reverse primer <400> 4 ttagtttttc agttgcactg ttaaatttcc 30

Claims (5)

(1) isolating RcLPAT376 (Castor bean lysophosphatidyl acyltransferase 376) gene from a castor ( Ricinus communis L.) using a primer set represented by SEQ ID NOS: 3 and 4;
(2) preparing a recombinant vector comprising the RcLPAT376 gene comprising the nucleotide sequence of SEQ ID NO: 1 isolated in the step (1);
(3) transforming the vector into a plant using Agrobacterium;
(4) screening the transgenic plants by spraying BASTA herbicide;
(5) obtaining T1 seeds from the selected transgenic plants;
(6) selecting a line in which the content of hydroxy fatty acid in the T1 seed of step (5) is increased;
(7) developing the system selected in step (6) to T2 generation;
(8) selecting a line in which the content of the hydroxy fatty acid is increased in the T2 seed of the step (7); And
(9) A method for producing a hydroxy fatty acid comprising the step of obtaining a hydroxy fatty acid from a seed of a T3 generation of the step (8)
Wherein the hydroxy fatty acid is 12-hydroxy-octadeca-9-enoic acid. 12. The method of claim 11, wherein the hydroxy fatty acid is 12-hydroxy-octadeca-9-enoic acid. The method according to claim 1, wherein the RcLPAT376 gene is expressed in male flower. 2. The method of claim 1, wherein the RcLPAT376 gene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 2. The method of claim 1, wherein the plant is a Arabidopsis or a vegetable oil plant. 5. The method of claim 4, wherein the preserved plant is selected from the group consisting of sesame, rapeseed, sunflower, castor, peanut, bean, coconut, oil palm, olive, soybean, corn and jojoba.

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