KR101399946B1 - Method for producing transgenic plant with increased syringin production and the plant thereof - Google Patents

Abstract

The present invention relates to recombinant herpes simplex mutant UGT72E3 / 2 gene having excellent synthase-synthesizing ability with excellent enzyme specificity for cinnamyl alcohol, F5H, CHS gene involved in phenylpropanoid biosynthetic pathway, positive gene for lignin biosynthesis pathway The present invention relates to a method for producing a transgenic plant having an increased production of syringine by a metabolic engineering method using a regulatory transcription factor Myb58 gene, It is expected to enable the development of high value-added agriculture biomaterials industry because it can mass-produce.

Description

FIELD OF THE INVENTION The present invention relates to a method for producing transgenic plants having increased production of syringine,

The present invention relates to a method for producing transgenic plants with increased production of syringine, and to plants therefrom. More particularly, the present invention relates to a recombinant vector comprising a gene encoding a recombinant herpes simplex UGT72E3 / 2 protein and a recombinant vector containing F5H (furulate 5- wherein the transformed plant is transformed with a recombinant vector comprising a gene coding for a hydroxylase protein to increase syringin production compared to the wild type and a method for producing the transgenic plant; A recombinant vector comprising a gene encoding a UGT72E3 / 2 protein, a recombinant vector comprising a gene encoding a F5H protein, and a gene encoding a Myb58 or Myb63 protein, Increased transgenic plants and methods of producing the transgenic plants; The recombinant vector was transformed into a silent vector that knock-out a recombinant vector containing the gene encoding the enzyme UGT72E3 / 2 protein and a gene encoding CHS (chalcone synthase) protein, resulting in increased production of syringine compared to the wild type Transformed plants and methods for producing the transgenic plants; To a transgenic plant having increased syringine production as compared to the wild type produced by each of the above methods and to seeds thereof.

Syringin is a lignin-based glycosaminoglycan, which is produced by the synthesis of phenylpropanoid, which is a component of lignin, . Therefore, in order to effectively produce the syringine through the phenylpropanoid synthesis pathway in the plant, it is necessary to have a specificity for the sinapyl alcohol precursor, sinapyl alcohol, There is a need for a metabolic engineering technique that can increase the content of existing cinephyl alcohol.

Stress is a source of panic. Chronic stress induces a variety of physiological and functional disorders and causes diseases such as neurogenic neurosis, depression, chronic fatigue, cardiovascular disease and metabolic disorders, as well as excessively persistent steroid hormones in the adrenal glands, . In particular, it has been reported that cancer inhibitory effect by cell-mediated immunological action by cytotoxic T-cells is weakened, and cancer cell growth is promoted. However, in today 's competitive social structure, it is impossible to live completely liberated from stress, so modern people are trying to enhance their adaptability to stress by reducing their stress. Research on adaptogen (Adaptogen), which is a natural pharmacological ingredient effective for improving mental, physical and adaptive capacity against stress, and industrial usability are being actively carried out.

An adaptogen is a plant secondary metabolite that responds to a variety of stresses and increases the non-specific resistance of the organism without adverse effects, thereby inducing adaptability to stress conditions. In general, extractive mixtures of Omija, Hongyeongcheon, and Asiatic plants, which have a large number of adaptation components, are traditionally used. In particular, it is famous for its function as a natural nourishing tonic which not only prevents aging but also stimulates appetite, strengthens energy and improves memory. As described above, it has been known that Gassiogapi is superior to ginseng and wild ginseng. Recently, efforts have been made to develop various health foods by using OGFP. However, only a liquid extract of OGFP or ogapiju . The study of the important chemical components of this pharmacological effect of Ganoderma Lucidum was studied in earnest in the 1970s with the use of Ganoderma Lucidum to overcome stress in Russian astronauts. The roots and shells of Liliaceae are used as medicinal materials. Elements of lignin complexes such as Eleutherosides A, B, C, D, K, L and M are important components. In particular, it has been clinically confirmed that Syringin and E (Acanthoside D) function very well.

Especially, it has been reported that Elletoroside B (chirinin) has excellent efficacy in the treatment of diabetes and depression, which are the most important health problems of modern urban people. However, there is a limited number of cultivated areas and there is a large difference in pharmacological composition according to cultivated areas. Therefore, it is necessary to develop stable production technology of plant secondary metabolites, which are high value-added products such as syringin, through the regulation of plant metabolic pathway using biotechnology.

Korean Patent Laid-Open Publication No. 2004-0004764 discloses a method for screening a compound of the present invention for preventing or inhibiting hepatotoxicity, Discloses a composition having an antioxidative action and hepatotoxic protecting activity containing a capyranoside, and Korean Patent Laid-Open No. 1998-0072707 discloses a pharmaceutical composition of a sirolin having a hepatic function. However, The production method of transgenic plants having an increased production of syringine and the plants therefrom have not been disclosed at all.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for regulating an essential step for regulating the flow of substrates in phenylpropanoid synthesis pathway, Transgenic plants overexpressing the genes and transformants deficient in the CHS gene function were prepared. The transgenic plants were transfected with the transformants overexpressing the recombinant glycoprotein UGT72E3 / 2, . As a result, it was confirmed that the production of silincin in the transgenic plants overexpressing UGT72E3 / 2 and F5H was significantly increased. In addition, the Arabidopsis transformants overexpressing Myb58, a positive regulatory transcription factor of the lignin synthesis pathway, were prepared and mated with transformants overexpressing UGT72E3 / 2 and F5H, The present inventors have completed the present invention by confirming that the production amount of silginin in the transgenic plants simultaneously overexpressing the UGT72E3 / 2, F5H, and Myb58 proteins was increased by at least 10 times as compared to when the UGT72E3 / 2 protein alone was overexpressed.

In order to solve the above problems, the present invention relates to a recombinant vector comprising a recombinant vector comprising a gene encoding a recombinant herpes virus UGT72E3 / 2 protein and a gene encoding a F5H (furulate 5-hydroxylase) Lt; RTI ID = 0.0 > syringin < / RTI > production.

The present invention also provides a recombinant vector comprising a recombinant vector comprising a gene encoding a recombinant herpes simplex protein UGT72E3 / 2 protein, a recombinant vector comprising a gene encoding a furulate 5-hydroxylase (F5H) protein, and a gene encoding Myb58 protein The present invention provides a transgenic plant which is transformed with a recombinant vector and has an increased production of syringine compared with the wild type.

The present invention also relates to a recombinant vector comprising a recombinant vector comprising a gene encoding a recombinant herpes virus UGT72E3 / 2 protein, a recombinant vector comprising a gene encoding a furulate 5-hydroxylase (F5H) protein, and a gene encoding Myb63 protein The present invention provides a transgenic plant which is transformed with a recombinant vector and has an increased production of syringine compared with the wild type.

Further, the present invention relates to a recombinant vector comprising a recombinant vector containing a gene encoding a recombinant herpes virus UGT72E3 / 2 and a silencing vector knocking out a gene encoding a CHS (chalcone synthase) protein, To provide transgenic plants with increased production of syringine.

In addition, the present invention relates to a method of screening for a transgenic plant which overexpresses UGT72E3 / 2 protein and F5H protein simultaneously by crossing the UGT72E3 / 2 protein overexpressing transgenic plant and the F5H protein overexpressing transgenic plant, The present invention provides a method for producing transgenic plants having increased production.

The present invention also provides a method for screening transgenic plants which simultaneously overexpress the UGT72E3 / 2 protein, the F5H protein and the Myb58 protein by crossing the transgenic plant overexpressing the UGT72E3 / 2 protein and the F5H protein and the Myb58 protein overexpressing transgenic plant The present invention provides a method for producing a transgenic plant having an increased production of syringine compared with a wild-type strain containing the step.

In addition, the present invention provides a method for screening transgenic plants which simultaneously overexpress the UGT72E3 / 2 protein, the F5H protein and the Myb63 protein by crossing the transgenic plants overexpressing the UGT72E3 / 2 protein and the F5H protein and the Myb63 protein overexpressing transgenic plant The present invention provides a method for producing a transgenic plant having an increased production of syringine compared with a wild-type strain containing the step.

In addition, the present invention includes a step of crossing a UGT72E3 / 2 protein overexpressed transgenic plant with a plant knocked out of a CHS protein coding gene to overexpress the UGT72E3 / 2 protein and selecting a transgenic plant in which the expression of CHS protein is inhibited The present invention provides a method for producing transgenic plants having an increased production of syringine compared to the wild type.

The present invention also provides a transgenic plant having increased production of syringine compared to the wild type produced by each of the above methods.

The present invention also provides a seed of the plant.

According to the present invention, sufficient production of cinnamyl alcohol, a precursor of syringine by metabolic engineering through F5H, CHS and Myb58 gene regulation involved in the phenylpropanoid synthesis pathway in plants, and recombinant glycosyltransferase enzyme UGT72E3 / 2 The synergistic effect by the enhancement of the sugar-active activity used enables the development of high value-added agricultural biomaterials industry by providing a new method to effectively mass-produce the pharmacologically highly purified syringine from various plants. .

FIG. 1 shows the results of a comparison of the glycosyltransferase activities in the Arabidopsis transformants and the protein extracts of wild-type leaves overexpressing the UGT72E2, UGT72E3, UGT72E2 / 3 and UGT72E3 / 2 genes, respectively. Coniferyl alcohol or cinapyl alcohol was added to the protein extract of the leaves of the plants and the resulting coniferin and syringine after 60 minutes of reaction were measured to indirectly measure the activity of the glycosyltransferase present in the protein extract of each transformant It is. (A) conipherin production, (B) silin production.
Fig. 2 shows the synthesis route of phenylpropanoid for the synthesis of syringin and the regulatory region of the gene used in the present invention.
FIG. 3 shows the results of RT-PCR of the expression level of each gene in Arabidopsis transformants overexpressing HCT, F5H and Myb58 genes. Actin 2 gene was used as a control.
FIG. 4 is a graph showing the synergistic effect of the transgene UGT72E3 / 2 gene and the HCT, F5H and CHS genes in the phenylpropanol synthesis pathway. In order to investigate the synergistic effect of the transgene UGT72E3 / 2 gene and the HCT, F5H and CHS genes in the phenylpropanol synthesis pathway, Lt; / RTI > shows the quantitative HPLC analysis of (A) and syringine (B) production. (C) Peak 1 of each chromatogram represents coniferyl alcohol 4-O-glucoside (coniferin), and peak 2 represents cinnamyl alcohol 4-O-glucoside (cyclinine).
FIG. 5 is a graph showing the synergistic effects of the transgene UGT72E3 / 2 gene and the HCT, F5H and CHS genes in the phenylpropanol synthesis pathway. In order to investigate the synergistic effects of the transgene UGT72E3 / 2 gene and the HCT, F5H and CHS genes in the phenylpropanol synthesis pathway, Lt; / RTI > shows the quantitative HPLC analysis of (A) and syringine (B) production. (C) Peak 1 of each chromatogram represents coniferyl alcohol 4-O-glucoside (coniferin), and peak 2 represents cinnamyl alcohol 4-O-glucoside (cyclinine).
FIG. 6 is a graph showing the results of the synergistic effect of the pyrolyzing of the F5H gene in the synthetic pathway of the UGT72E3 / 2 gene and the phenylpropanol synthesis pathway and the Myb58 gene, which is a positive regulatory factor of the genes involved in the lignin synthesis pathway, The quantitative HPLC analysis shows that the production of conipiperin (A) and cyclin G (B) is remarkably increased. (C) Peak 1 of each chromatogram represents coniferyl alcohol 4-O-glucoside (coniferin), and peak 2 represents cinnamyl alcohol 4-O-glucoside (cyclinine).
FIG. 7 shows the synergistic effect of the accumulation of Myb58 gene, which is a positive regulatory transcription factor of the F5H gene and the gene involved in the lignin synthesis pathway, in the synthesis pathway of the UGT72E3 / 2 gene and the phenylpropanol complex, The results are shown by HPLC analysis. (C) Peak 1 of each chromatogram represents coniferyl alcohol 4-O-glucoside (coniferin), and peak 2 represents cinnamyl alcohol 4-O-glucoside (cyclinine).

In order to accomplish the object of the present invention, the present invention provides a recombinant vector comprising the gene encoding the recombinant glycoprotein UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 and F5H (furulate 5 -hydroxylase < / RTI > protein, thereby increasing syringin production as compared to the wild type.

The present invention also provides a recombinant vector comprising a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2, a furulate 5-hydroxylase (F5H) protein comprising the amino acid sequence of SEQ ID NO: 4 And a recombinant vector comprising a gene encoding Myb58 protein consisting of the amino acid sequence of SEQ ID NO: 6, thereby increasing the production of syringine compared to the wild type.

The present invention also provides a recombinant vector comprising a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2, a furulate 5-hydroxylase (F5H) protein comprising the amino acid sequence of SEQ ID NO: 4 A recombinant vector comprising a gene encoding Myb63 protein consisting of the amino acid sequence of SEQ ID NO: 8, and a recombinant vector comprising a gene encoding Myb63 protein comprising the amino acid sequence of SEQ ID NO: 8, thereby increasing the production of syringine compared to the wild type.

The present invention also provides a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 and a gene encoding a CHS (chalcone synthase) protein consisting of the amino acid sequence of SEQ ID NO: 10 Provides a transgenic plant that is transformed into a silent vector that knock-out, resulting in an increased production of syringine compared to the wild type.

UGT72E2, a UDP-dependent glucosyltransferase (UGT72E2) isolated from Arabidopsis thaliana in the prior art, has a very high glycosyltransferase efficiency but has a weak substrate specificity for coniferyl alcohol, whereas the enzyme UGT72E3 In order to overcome this disadvantage, it was found that the substrate specificity was strongly maintained and a new recombinant sugar complex with enhanced sugar chain activity was obtained. To prepare the enzyme, the recombinant genes UGT72E2 / 3 and UGT72E3 / 2 were constructed using domain swapping method from UGT72E2 and UGT72E3 genes.

Specifically, in the present invention, the UGT72E2 and UGT72E3 genes were divided into carboxy fragments containing an amino fragment containing amino acids 1 to 344 of amino terminal and an amino acid of amino acid number 345 to 481 of carboxy terminal, respectively. The amino fragment contains a region that determines the substrate recognition specificity and the carboxy terminal contains PSPG motifs important for glycosylation. In order to efficiently produce the syringine in the plant, the substrate specificity is more important than the glycoprotein activity. Therefore, the amino fragment is enlarged to about 3/4 of the whole, and the carboxy fragment is divided into the minimum size including the PSPG motif.

The recombinant UGT72E2 / 3 gene used in the present invention encodes an amino fragment containing amino acids 1 to 344 at the amino terminus of UGT72E2 and a carboxy fragment encoding the carboxy fragment containing amino acids 345 to 481 of carboxy terminal of UGT72E3 The recombinant UGT72E3 / 2 gene was prepared by linking an amino fragment containing the amino acids 1 to 344 of the amino terminal of UGT72E3 and a carboxy fragment containing amino acids 345 to 481 of the carboxy terminal of UGT72E2 Were linked to each other.

To investigate the enzymatic properties of the recombinant genes, Arabidopsis thaliana transformants overexpressing UGT72E2, UGT72E3, UGT72E2 / 3 and UGT72E3 / 2 genes were prepared using Agrobacterium, As a result of the comparison, it was confirmed that the recombinant glycosyltransferase UGT72E3 / 2, which was newly produced, was significantly increased in the synthesis of syringin compared to the wild type. In the present invention, UGT72E3 / 2 was used to produce high- .

In the present invention, in order to efficiently supply cinnamyl alcohol, which is a substrate of the serine, to the transformants overexpressing the recombinant herbal transformant UGT72E3 / 2, the respective steps and action enzymes of the serine synthase pathway were used. Coumaryl- Over-expression of HCT (hydroxycinamoyl-CoA: shikimate / quinquat hydroxycinamoyl transferase (HCT) gene) to reduce the amount of coniferyl aldehyde converted to coniferyl alcohol and conversion to cinnamal alcohol F5H (ferulate 5-hydroxylase) gene was overexpressed in order to stimulate the immune response. In addition, mutants deficient in chalcone synthase (CHS) gene function were used to reduce the amount of coumaryl-CoA escaping into the flavonoid pathway in the synthesizing pathway. A silencing vector may be used to knock out the CHS gene, but is not limited thereto.

The term "knock-out" of the present invention means modification or elimination of a specific gene in a nucleotide sequence so that it can not be expressed. Generally, the expression of a gene is downregulated or completely suppressed .

The term "recombinant" of the present invention refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The recombinant plant expression vector of the present invention can be used as a transient expression vector capable of transient expression in a plant into which the foreign gene is introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant.

Binary vectors that can be used in the present invention include the RB (right border) and LB (left border) of T-DNA, which can transform a plant when present together with the Ti plasmid of Agrobacterium tumefaciens (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vector, etc., which are frequently used in the art, may be used good.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "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.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. Particularly preferred forms of the Ti-plasmid vector are described in EP 0 120 516 B1 and US Patent 4,940,838, It is a vector. Other suitable vectors that can be used to introduce a gene according to the invention into a 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 expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistant genes such as kanamycin, Ampicillin, G418, Bleomycin, hygromycin, chloramphenicol, Genes, but are not limited thereto.

In a plant expression vector according to an embodiment of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the 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 plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In a plant expression vector according to an embodiment of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agro And the terminator of the Octopine gene of Agrobacterium tumefaciens , but is not limited thereto.

The present invention also relates to

(a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;

(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant; And

(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) Wherein the syringin production is increased as compared to the wild type, wherein the syringin production is increased.

The present invention also relates to

(a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;

(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant;

(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) step;

(d) transforming a plant with a recombinant vector comprising a gene encoding Myb58 protein consisting of the amino acid sequence of SEQ ID NO: 6 to produce a Myb58 protein over-transgenic plant;

(e) crossing the transgenic plants overexpressing the selected UGT72E3 / 2 protein and F5H protein of step (c) and the Myb58 protein overexpressing transgenic plant of step (d) to produce UGT72E3 / 2 protein, F5H protein and Myb58 A method for producing a transgenic plant having increased syringin production as compared to a wild-type plant, which comprises producing a transgenic plant that overexpresses a protein at the same time.

The present invention also relates to

(a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;

(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant;

(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) step;

(d) transforming a plant with a recombinant vector comprising a gene encoding Myb63 protein consisting of the amino acid sequence of SEQ ID NO: 8 to produce Myb63 protein over-transgenic plant;

(e) crossing the transgenic plants overexpressing the selected UGT72E3 / 2 protein and the F5H protein of step (c) and the Myb63 protein overexpressing transgenic plant of step (d) to produce UGT72E3 / 2 protein, F5H protein and Myb63 A method for producing a transgenic plant having increased syringin production as compared to a wild-type plant, which comprises producing a transgenic plant that overexpresses a protein at the same time.

The present invention also relates to

(a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;

(b) preparing a knock-out plant in which a gene encoding a chalcone synthase (CHS) protein consisting of the amino acid sequence of SEQ ID NO: 10 is knocked out; And

(c) transfecting the UGT72E3 / 2 protein-overexpressed transgenic plant of step (a) with a plant knocked out of the CHS protein coding gene of step (b) to overexpress the UGT72E3 / 2 protein, The present invention provides a method for producing a transgenic plant having increased syringin production as compared to the wild type, comprising the step of selecting a transformed plant.

In the method according to one embodiment of the present invention, the plant is preferably, but not limited to, increased syringin synthesis in the leaves or roots, and most preferably increased syringin synthesis in the leaves.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

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

The Myb58 gene used in the present invention is a transcription factor that positively regulates the genes involved in the phenylpropanoid biosynthesis pathway, and Myb63, which is known to perform similar functions, is also expected to produce a synergistic effect in the production of syringine.

The present invention also provides transgenic plants and seeds thereof having increased syringine production compared to the wild type produced by each of the above methods.

In a plant according to an embodiment of the present invention, the plant is preferably selected from the group consisting of Arabidopsis thaliana, tobacco, eggplant, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, And may be a dicot which is a Chinese cabbage, a cabbage, a Chinese cabbage, a watermelon, a melon, a cucumber pumpkin, a pak, a strawberry, a soybean, a mung bean, a kidney bean and a pea.

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

The gene and the base sequence information used in the present invention

The gene and nucleotide sequence information used in the present invention are shown in Table 1 below.

Gene name Base sequence number Amino acid sequence number UGT72E3 / 2 One 2 F5H 3 4 Myb58 5 6 Myb63 7 8 CHS 9 10 HCT 11 12

Example  One. The party  enzyme UGT72E2 , UGT72E3 , UGT72E2 / 3, UGT72E3 / 2 < / RTI > genes and the protein extracts prepared from the wild-type leaves The party  Comparison of enzymes activity

In general, most of the monolignol produced by the activation of the phenylpropanoid synthesis pathway in the seedlings leaves coniferyl alcohol converted to conipherin and only a few coniferyl alcohols are converted to F5H (furulate 5-hydroxylase), COMT (caffeic acid 3-O-methyltransferase), and CAD, to the sinapine alcohol, a precursor of syringine. Therefore, in addition to the enzyme activity, the supply of cinnamaldehyde, a substrate, is an important determinant of syringine production.

In order to compare the activities of these enzymes, Arabidopsis transformants overexpressing the UGT72E2, UGT72E3, UGT72E3, UGT72E2 / 3 and UGT72E3 / 2 genes, respectively, and protein extracts prepared from wild-type leaves were used as substrates. Coniferyl alcohol or cinnamyl 1 mM of alcohol and 5 mM of UDP-glucose were added and reacted at 22 DEG C for 60 minutes. Thereafter, the reaction was stopped by adding methanol twice by volume to the reaction solution, and the amounts of conipherin and silinene produced before and after the reaction were quantified by HPLC. I already have biometric (in vivo ), the recombinant herbal enzyme UGT72E3 / 2 had a strong substrate specificity for cinnamyl alcohol and a high glycosyltransferase activity. In particular, UGT72E3 / 2 showed a higher rate of production of syringine as compared to the other glycosylated enzymes (Fig. 1).

The above results indicate that the use of a method of increasing the supply of cyphenyl alcohol by a metabolic engineering method to a transformant overexpressing UGT72E3 / 2 is capable of producing high efficiency syringine in plants.

Example  2. For synthesis of syringin Phenylpropanoid  Synthetic pathway and gene control site

In order to efficiently supply cinnamyl alcohol, which is a substrate of serine, to the transformants overexpressing the recombinant glycoprotein UGT72E3 / 2 by metabolic engineering, each step of the phenylpropanoid synthesis pathway and its action enzymes were used. Mutants deficient in the chalcone synthase (CHS) gene were used to reduce the amount of coumaryl-CoA escaping into the flavonoid pathway in the synthetic pathway. In addition, in order to enhance the entry of the phenylpropanoid synthesis pathway of coumaryl-CoA, overexpression of HCT (hydroxycinamoyl-CoA: shikimate / quinquat hydroxycinamoyl transferase) gene, reduction of the amount of coniferyl aldehyde converted to coniferyl alcohol, A strategy of overexpressing the F5H (ferulate 5-hydroxylase) gene was used to promote the conversion to alcohol (Fig. 2).

Example  3. Phenylpropanoid  Synthetic pathway control gene HCT , F5H and Myb58  Production of overexpressed Arabidopsis transformants and confirmation of the expression level of each gene

Binary vectors were prepared so that the Arabidopsis HCT, F5H and Myb58 gene coding regions were regulated by the super-promoter, and then the vector was transformed into Agrobacterium tumefaciens ) EHA105 and then transformed into Arabidopsis by in planta method using the bacteria. At this time, kanamycin (HCT and F5H) or herbicide (Myb58) was added to a UGT72E3 / 2 overexpressed transformant previously prepared with a hygromycin resistance selection marker in order to accumulate the HCT, F5H and Myb58 genes in a pyramidal manner through a cross- ) Resistance selection markers were used to construct Arabidopsis transformants.

Since the phenotype of the transformant is greatly influenced by the expression level of the introduced transgene, the amount of the HCT, F5H and Myb58 gene inserted and expressed in the genome of the selected transformants was determined by RT-PCR Respectively. (5'-CTGGTTACTTTGGGAATGTGATATTCAC-3 '; SEQ ID NO: 13), F5H gene-specific forward primer F5H-F (5' (SEQ ID NO: 14) and Myb58 gene specific forward primer Myb58-F (5'-CAGACGAGTTGAAGAATCCGACATCGAG-3 '; SEQ ID NO: 15) were used as reverse primers. Specific primers for the 3' UTR region of the vector The primer UTR-R (5'-TTAAAGCAGGGCATGCCTGC-3 '; SEQ ID NO: 16) was used. To compensate for the relative amount of RNA, the Arabidopsis thaliana actin 2 gene, which is constantly expressed, was used as a reference gene. Ten transgenic lines were examined for each gene, and a transformant line excellent in HCT, F5H and Myb58 gene expression was finally obtained (FIG. 3). In addition, there is only one CHS gene in Arabidopsis, and when the gene is defective, the color of the seed shell changes to yellow. These phenotypic differences were used to isolate the pure transformants.

Example  4. In the leaves and roots of transformants Connie Perrin and  Quantitative HPLC analysis of syringine formation

Seedlings produce three types of monolignol through the phenylpropanoid synthesis pathway: H monolignol using p-coumaryl alcohol, G monolignol using coniferyl alcohol and S monoligol using cinapyl alcohol are produced. However, the concentration of coniferyl alcohol in plant cells is relatively high because most of them are in the form of G mono-lignol. Overexpression of this enzyme, UGT72E2, converts high concentrations of coniferyl alcohol into conipherin. Concentrations of cinnamic alcohol, a precursor of syringes in plant nests, are very low because some coniferyl alcohol is converted to cinnamic alcohol by the enzymatic action of F5H and COMT (caffeic acid 3-O-methyltransferase). In order to produce high-efficiency syringine in plants, metabolic engineering is needed to increase the concentration of cinnamaldehyde in combination with the highly efficient glycated enzyme UGT72E3 / 2.

Quantitative HPLC analysis of coniferin and sulindin formation in the leaves and roots of transformants was performed to investigate the synergistic effects of the transgene UGT72E3 / 2 gene and the HCT, F5H and CHS genes of phenylpropanoid. Transformants overexpressing HCT and F5H genes, which regulate important steps of the phenylpropanol synthesis pathway, were prepared and crossed with transgenic plants overexpressing the high-efficiency glycosyltransferase gene UGT72E3 / 2. Subsequently, transgenic plants overexpressing HCT and UGT72E3 / 2 or F5H and UGT72E3 / 2 genes were separately isolated in the F2 generation, and a pure line was obtained in the next generation. In addition, chalcone synthase (CHS), which converts p-coumaryl-CoA to chalcone, is defective to prevent p-coumaryl-CoA, an important precursor of the phenylpropanoid pathway, from escaping into the flavonoid synthesis pathway Mutant and a transgenic overexpressing UGT72E3 / 2, followed by isolation of a line in which the CHS gene is knocked out in the F2 generation and the UGT72E3 / 2 gene is overexpressed, followed by obtention in the next generation Respectively.

As a result of the synthesis efficiency of the syringine by HPLC in the leaves and roots of the transformants, it was found that the plant line with the overexpression of the HCT or the F5H gene and the CHS gene function deficiency, compared to the transformant alone overexpressing the UGT72E3 / , The synthesis of syringine was increased by 17.3%, 71.3% and 64.6%, respectively (Fig. 4).

However, in the roots, the synthesis of the syringin was slightly increased only in the transformant line overexpressing the UGT72E3 / 2 and F5H genes (Fig. 5), compared with the UGT72E3 / 2 gene alone overexpressing the transformant.

Example  5. UGT72E3 / 2, F5H, and Myb58  Gene Pyramid  Quantitative Analysis of the Formation of Syringine in the Leaf and Root of the Transformant by Synergy Effect HPLC  analysis

The accumulation of overexpression of the F5H gene, which is involved in the conversion of a novel glycosyltransferase gene UGT72E3 / 2 and coniferyl alcohol in the pathway of phenylpropanol to cinnamyl alcohol, which was developed by an enzymatic engineering method, The production rate was significantly increased. However, when plant roots are exposed to light, the expression of various genes involved in the phenylpropanoid synthesis pathway is increased by the light signaling mechanism. As a result, the amount of synthesized syringine in wild type roots was more than 36 times higher than that of leaves, because the synthesis of monoriginal containing a large amount of coniferyl alcohol and cinapyl alcohol was increased. In the leaves of transgenic plants overexpressing UGT72E3 / 2 and F5H genes, the amount of silginin was 16 times higher than that of wild-type leaves. However, the amount of silginin produced in the root of transformants was still 4 times Respectively. Increasing the yield of syringine from the leaves has the advantage that it can be mass-grown without destroying the plant, unlike the root. Therefore, it is necessary to increase the expression of many genes involved in the phenylpropanoid pathway, like roots exposed to light in leaves.

In order to solve the above problem, the present invention provides a synergistic effect using pyramidation of the Myc58 gene, which is a positive regulatory transcription factor of the gene encoding the transgene UGT72E3 / 2 gene, the F5H gene of the phenylpropanoid synthesis pathway and the gene involved in the lignin synthesis pathway Was determined by quantitative HPLC analysis.

In order to confirm the synergistic effect of the Myb58 gene, a transformant overexpressing the Myb58 gene, which is a transcription factor that specifically regulates the lignin biosynthesis pathway in Arabidopsis thaliana, was prepared. Since the Myb58 gene can not improve the expression of the F5H gene, the transformant line overexpressing all the Myb58, UGT72E3 / 2 and F5H genes in the F2 generation is selected by crossing with the transformant overexpressing both the UGT72E3 / 2 and the F5H gene Obtained obedience in the next generation. As a result of HPLC analysis of the synthesis efficiency of syringine in the leaves and roots of these transformants, the yield of silginin in the leaves of transformants overexpressing UGT72E3 / 2, F5H and Myb58 genes was found to be higher than that of UGT72E3 / 2 and F5H 8-fold and UGT72E3 / 2 alone, respectively, compared to the transgenic overexpressed transgenic plants. Furthermore, the yield of the syringine in the leaves of the transformants was found to be increased about twice as much as that in the roots. As a synergistic effect of the overexpression of UGT72E3 / 2, F5H and Myb58 genes, it was found that ideally the production of the syringine in the root was doubled and the production of syringine in the leaves was greatly increased. Mass-producing plant transformants were completed.

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Claims (12)

A recombinant vector comprising the gene encoding the recombinant glycosyltransferase UGT72E3 / 2 protein consisting of the amino acid sequence of SEQ ID NO: 2, and a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: Transgenic plants that have been transformed with the vector and have increased syringin production relative to the wild type. A recombinant vector comprising the gene encoding the recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: And a gene encoding Myb58 protein consisting of the amino acid sequence of SEQ ID NO: 6, thereby increasing the production of syringine compared to the wild-type. A recombinant vector comprising the gene encoding the recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: And a gene encoding Myb63 protein consisting of the amino acid sequence of SEQ ID NO: 8, thereby increasing the production of syringine compared to the wild-type. A recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 and a gene encoding a CHS (chalcone synthase) protein consisting of the amino acid sequence of SEQ ID NO: 10 were knocked out ) With increased silencing production compared to the wild type. (a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;
(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant; And
(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) Wherein the syringin production is increased compared to the wild type, wherein the syringin production is increased. (a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;
(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant;
(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) step;
(d) transforming a plant with a recombinant vector comprising a gene encoding a Myb58 protein of SEQ ID NO: 6 to produce a Myb58 protein-overexpressing transgenic plant;
(e) crossing the transgenic plants overexpressing the selected UGT72E3 / 2 protein and F5H protein of step (c) and the Myb58 protein overexpressing transgenic plant of step (d) to produce UGT72E3 / 2 protein, F5H protein and Myb58 Wherein the syringin production is increased compared to the wild type, wherein the syringin production is increased as compared to the wild type. (a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;
(b) transforming a plant with a recombinant vector comprising a gene encoding an F5H (furulate 5-hydroxylase) protein consisting of the amino acid sequence of SEQ ID NO: 4 to produce an F5H protein overexpressing transgenic plant;
(c) selecting transgenic plants that overexpress the UGT72E3 / 2 protein and the F5H protein by crossing the UGT72E3 / 2 protein overexpressed transgenic plant of step (a) with the F5H protein overexpressed transgenic plant of step (b) step;
(d) transforming a plant with a recombinant vector comprising a gene encoding Myb63 protein consisting of the amino acid sequence of SEQ ID NO: 8 to produce Myb63 protein over-transgenic plant;
(e) crossing the transgenic plants overexpressing the selected UGT72E3 / 2 protein and the F5H protein of step (c) and the Myb63 protein overexpressing transgenic plant of step (d) to produce UGT72E3 / 2 protein, F5H protein and Myb63 Wherein the syringin production is increased compared to the wild type, wherein the syringin production is increased as compared to the wild type. (a) transforming a plant with a recombinant vector comprising a gene encoding a recombinant glycosyltransferase UGT72E3 / 2 protein comprising the amino acid sequence of SEQ ID NO: 2 to produce a transgenic plant transfected with UGT72E3 / 2 protein;
(b) preparing a knock-out plant in which a gene encoding a chalcone synthase (CHS) protein consisting of the amino acid sequence of SEQ ID NO: 10 is knocked out; And
(c) transfecting the UGT72E3 / 2 protein-overexpressed transgenic plant of step (a) with a plant knocked out of the CHS protein coding gene of step (b) to overexpress the UGT72E3 / 2 protein, A method for producing a transgenic plant having increased syringin production as compared to a wild type, comprising the step of selecting a transformed plant. 9. A transgenic plant having increased syringin production compared to the wild type produced by the method of any one of claims 5-8. 10. The transgenic plant according to claim 9, wherein the plant is a dicotyledonous plant. 10. The transgenic plant according to claim 9, wherein the plant is a Arabidopsis thaliana. 10. A seed of a transgenic plant according to claim 9.

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