# Patent

## Title
Method for producing transgenic plant with increase syringin production and plant produced by using the same

## Abstract

The present invention relates to recombinant glycosyl transferase UGT72E3/2 gene having an excellent syringin synthesis ability based on remarkable enzyme specificity for sinapyl alcohol, a method for producing a transgenic plant with increased syringin production based on a metabolic process which uses F5H and CHS genes that are involved with the phenylpropanoid biosynthesis pathway and Myb58 gene as a transcription factor for positive regulation of the gene that is involved with lignin biosynthesis pathway, and a plant obtained by the method. According to the present invention, syringin with various pharmaceutical applications can be effectively produced in a large amount in a plant, and thus it is expected to allow the development of an industry relating to agrobiological materials that are highly valuable as foods or pharmaceuticals.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY
This patent application claims benefit under 35 U.S.C. 119(e), 120, 121, or 365(c), and is a National Stage entry from International Application No. PCT/KR2012/010856, filed Dec. 13, 2012, which claims priority to Korean Patent Application Nos. 10-2012-0112763, filed Oct. 11, 2012, and 10-2012-0124906, filed Nov. 6, 2012, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to recombinant glycosyl transferase UGT72E3/2 gene having excellent syringin synthesis activity based on remarkable enzyme specificity for sinapyl alcohol, a method for producing a transgenic plant with increased syringin production based on metabolic engineering with F5H, HCT and CHS genes that are involved with the phenylpropanoid biosynthesis pathway and Myb58 gene as a transcription factor for positive regulation of the genes that are involved with lignin biosynthesis pathway, and a plant obtained by the method.
BACKGROUND ART
From sinapyl alcohol (s type monolignol) as a lignin-constituting component that is generated via phenylpropanoid synthesis pathway, syringin as s type monolignol glycoside is produced by glycosyl transferase (UDP-glucose transferase). In Arabidopsis thaliana as a model plant, there are about 100 kinds of glycosyl transferase, and it has been reported that, by forming glycosides of various compounds including plant hormones and secondary metabolites, they regulate the cellular activity and the storage of those compounds in a vacuole. Among them, UGT72E2 and UGT72E3 are reported as a glycosyl transferase responsible for converting firstly monolignol such as coniferyl alcohol and sinapyl alcohol specifically into monolignol-glycosides. Particularly, UGT72E3 as a sinapyl alcohol-specific glycosyl transferase required for syringin production has excellent substrate specificity for sinapyl alcohol, but its glycosyl transferase activity is so low that its application has been very limited. As such, development of a new glycosyl transferase useful for efficient production of syringin in a plant has been remained as a problem to be solved first before mass production and application of syringin, which is a pharmaceutically functional secondary metabolite.
Furthermore, for effective production of syringin in a plant via the phenylpropanoid synthesis pathway, it is also required to have a metabolic engineering technique which increase the content of sinapyl alcohol as a precursor of syringin that is present in a trace amount in a plant cell, as well as a strong glycosyl transferase activity having substrate specificity for sinapyl alcohol.
In particular, eleutheroside B (syringin) is classified as a representative adaptogen derived from plants, which is a pharmaceutical component from E. senticosus having excellent efficacy of psychological and physical adaptation against stress. The adaptogen is a terminology indicating a plant secondary metabolite which enhances non-specific resistance of a living body in response to various stresses without causing a side effect. Recently, syringin isolated in the pure state is reported to exhibit an excellent effect for diabetes and depression that become a serious problems of people living in modern-day cities, and thus its application is now broadened more than ever. However, because the area of cultivating E. senticosus is limited and there is a huge variation in pharmaceutical components depending on the area of cultivation, it is difficult to have the stable supply of E. senticosus required for production of syringin for commercial use. As such, development of a technique for stable production of a plant secondary metabolite like syringin, which is a highly valuable product, based on regulation of plant metabolic pathways using bioengineering techniques is needed.
Meanwhile, in Korean Patent Application Publication No. 2004-0004764, “Composition comprising extract of E. senticosus having hepato-protective activity or butyl alcohol soluble fraction thereof and butyl alcohol fraction of syringin and syringaresinol-di-O-β-D-glucopyranoside derivatives having anti-oxidant and hepato-protective activity” is disclosed. Further, in Korean Patent Application Publication No. 1998-0072707, “Pharmaceutical composition of syringin having liver function protecting activity” is disclosed. However, the method for producing a transgenic plant with increased syringin production and a plant obtained therefrom that are disclosed by the present invention have never been described before.
SUMMARY
The present invention is devised in view of the aforementioned needs, and to produce a new recombinant glycosyl transferase having enhanced glycosyl transfer activity while maintaining the substrate specificity at a high level. The recombinant gene UGT72E2/3 and UGT72E3/2 are produced by a domain swapping of UGT72E2 and UGT72E3 genes. Transgenic Arabidopsis thaliana over-expressing each of UGT72E family are generated with floral dipping method. Further, as a result of quantitative analysis of the efficiency for syringin synthesis, it is confirmed the newly produced recombinant glycosyl transferase UGT72E3/2 exhibits a significantly increased syringin synthesis than the wild type UGT72E2 and UGT72E3.
Further, in order to increase the syringin production via regulation of the metabolic pathway of a plant, each of the Arabidopsis thaliana transgenic plants over-expressing F5H and HCT genes, which regulate an important step for regulating the flow of substrates in the phenylpropanoid synthesis pathway, the transgenic plant deficient of CHS gene function, and the transgenic plant over-expressing Myb58 as a positive regulation transcription factor of the lignin synthesis pathway was produced. As a result of quantitative analysis of the efficiency for syringin synthesis after cross-breeding the aforementioned transgenic plants with the transgenic plant which over-expresses the recombinant glycosyl transferase UGT72E3/2, it was found that, from the newly produced transgenic plant which over-expresses simultaneously the proteins of UGT72E3/2, F5H, and Myb58, the production amount of syringin is increased by 10 times or more compared to the case in which only the UGT72E3/2 protein is over-expressed, and the present invention is completed accordingly.
To solve the problems described above, the present invention provides the recombinant glycosyl transferase UGT72E3/2 protein consisting of the amino acid sequence of SEQ ID NO: 2.
The present invention further provides the gene encoding the UGT72E3/2 protein.
The present invention further provides a recombinant vector comprising the gene encoding the UGT72E3/2 protein.
The present invention further provides a host cell transformed with the recombinant vector.
The present invention further provides a method of increasing syringin synthesis in a plant compared to the wild type, comprising transforming a plant cell with the recombinant vector to over-express the UGT72E3/2 gene.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein and a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein, and a recombinant vector comprising the gene encoding the Myb58 or Myb63 protein.
The present invention further provides a transgenic plant with increased syringin production in a plant compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein and a recombinant vector knock-outing the gene encoding the CHS (chalcone synthase) protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the UGT72E3/2 protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising cross-breeding a transgenic plant which over-expresses the UGT72E3/2 protein and a transgenic plant which over-expresses the F5H protein and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising cross-breeding a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein and a transgenic plant which over-expresses the Myb58 or Myb63 protein and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein, F5H protein, and Myb58 or Myb63 protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising cross-breeding a transgenic plant which over-expresses the UGT72E3/2 protein and a plant in which the gene encoding the CHS protein is knocked out and selecting a transgenic plant which over-expresses the UGT72E3/2 but has inhibited expression of the CHS protein.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, which is produced by each method described above, and a seed thereof.
The present invention still further provides a composition for increasing syringin synthesis in a plant comprising, as an effective component, a recombinant vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 1 which encodes the UGT72E3/2 protein.
According to the present invention, a new method for efficient and mass production of syringin in various plants, which is derived from a plant and has a widening application as it is reported to exhibit an excellent effect for treating diabetes and depression which become serious problems of people living in modern-day cities, is provided, by which pharmaceutically very useful syringin having a synergistic effect based on sufficient production of sinapyl alcohol, that is a precursor of syringin, and enhancement of the glycosyl transfer activity using the recombinant glycosyl transferase UGT72E3/2 using metabolic engineering via regulation of the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein and the F5H, CHS and Myb58 gene involved with phenylpropanoid synthesis pathway in a plant can be produced efficiently in large scale in various plants. Accordingly, it is expected to allow the development of an industry relating to agrobiological materials that are highly valuable as foods or pharmaceuticals.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing illustrating the comparison of the primary and secondary structures of Arabidopsis thaliana glycosyl transferase UGT72E2 and UGT72E3.
FIG. 2 is a drawing illustrating the comparison of the tertiary structure of glycosyl transferase UGT72B1(A) and VvGT1(B) that are derived from Arabidopsis thaliana or grape, glycosyl transferase UGT72E2 and UGT72E3 that are derived from Arabidopsis thaliana, and the recombinant glycosyl transferase UGT72E2/3 and UGT72E3/2(C) which have been produced in the present invention.
FIG. 3 is a drawing illustrating (A) the recombinant vector used for production of the transformant of the present invention, (B) expression level of the gene transferred in the transformant, and (C) reactivity in the transformed leaves against ultraviolet rays.
FIG. 4 is a drawing illustrating the quantitative HPLC analysis of the production of coniferin and syringin in leaves of the wild type Arabidopsis thaliana and the transformants, which express each of the four glycosyl transferase genes, i.e., UGT72E2, UGT72E3, UGT72E2/3, and UGT72E3/2 (peak of each chromatogram in (C): 1, coniferyl alcohol 4-O-glucoside (coniferin); 2, sinapyl alcohol 4-O-glucoside (syringin); 3, coniferyl alcohol; and 4, sinapyl alcohol).
FIG. 5 is a drawing illustrating the quantitative HPLC analysis of the production of coniferin and syringin in roots of the wild type Arabidopsis thaliana and the transformants, which express each of the four glycosyl transferase genes, i.e., UGT72E2, UGT72E3, UGT72E2/3, and UGT72E3/2 (peak of each chromatogram in (C): 1, coniferyl alcohol 4-O-glucoside (1, coniferyl alcohol 4-O-glucoside (coniferin); 2, sinapyl alcohol 4-O-glucoside (syringin); 3, coniferyl alcohol; and 4, sinapyl alcohol).
FIG. 6 is a drawing illustrating the nucleotide sequence and amino acid sequence of the recombinant glycosyl transferase UGT72E3/2.
FIG. 7 is a drawing illustrating the results of comparing the activity of glycosyl transferase, which is present in a protein extract prepared from the leaves of the Arabidopsis thaliana transformant over-expressing each of the glycosyl transferase UGT72E2, UGT72E3, UGT72E2/3 and UGT72E3/2, or the wild type, in which the activity of the glycosyl transferase present in the protein extract of each transformant was indirectly measured by determining coniferin and syringin, that are produced 60 minutes after adding coniferyl alcohol or sinapyl alcohol to the protein extract of the leaves of the transgenic plant. (A) Production amount of coniferin and (B) production amount of syringin.
FIG. 8 is a drawing illustrating the phenylpropanoid synthesis pathway for synthesis of syringin and the regulation site of the gene that is used in the present invention.
FIG. 9 illustrates the result of determining the expression amount of each gene in the Arabidopsis thaliana, which over-expresses each of the HCT, F5H and Myb58, based on RT-PCR. As a control group, Actin 2 gene was used.
FIG. 10 is a drawing illustrating the quantitative HPLC analysis of the production of (A) coniferin and (B) syringin in leaves of the transformants including wild type, which express various combinations of the genes, for determining the synergistic effect between the glycosyl transferase UGT72E3/2 and the HCT, F5H and CHS genes of the synthesis pathway for phenylpropanoid. Peak 1 of each chromatogram in (C) indicates coniferyl alcohol 4-O-glucoside (coniferin) and peak 2 indicates sinapyl alcohol 4-O-glucoside (syringin).
FIG. 11 is a drawing illustrating the quantitative HPLC analysis of the production of (A) coniferin and (B) syringin in roots of the transformants including wild type, which express various combinations of the genes, for determining the synergistic effect between the glycosyl transferase UGT72E3/2 and the HCT, F5H and CHS genes of the synthesis pathway for phenylpropanoid. Peak 1 of each chromatogram in (C) indicates coniferyl alcohol 4-O-glucoside (coniferin) and peak 2 indicates sinapyl alcohol 4-O-glucoside (syringin).
FIG. 12 is a drawing illustrating the quantitative HPLC analysis which shows a dramatic increase in the production of (A) coniferin and (B) syringin in the transformant leaves as caused by the synergistic effect based on pyramiding of the glycosyl transferase UGT72E3/2, the F5H gene in the synthesis pathway for phenylpropanoid, and the Myb58 gene as a transcription factor for positive regulation of the genes involved with the synthesis pathway for lignin. Peak 1 of each chromatogram in (C) indicates coniferyl alcohol 4-O-glucoside (coniferin) and peak 2 indicates sinapyl alcohol 4-O-glucoside (syringin).
FIG. 13 is a drawing illustrating the result of HPLC analysis which shows that the synergistic effect based on accumulation of the glycosyl transferase UGT72E3/2, the F5H gene in the synthesis pathway for phenylpropanoid, and the Myb58 gene as a transcription factor for positive regulation of the genes involved with the synthesis pathway for lignin has no significant effect in the roots of the transformant. Peak 1 of each chromatogram in (C) indicates coniferyl alcohol 4-O-glucoside (coniferin) and peak 2 indicates sinapyl alcohol 4-O-glucoside (syringin).
FIG. 14 is a drawing illustrating the expression amount of various genes involved with the synthesis pathway for phenylpropanoid in the transgenic Arabidopsis thaliana which over-expresses UGT72E3/2, F5H and Myb58 genes, the transgenic Arabidopsis thaliana which over-expresses UGT72E3/2 and F5H genes, and the wild type Arabidopsis thaliana as determined by RT-PCR. As a control group, Actin 2 gene was used.



DETAILED DESCRIPTION
In order to achieve the object described above, the present invention provides the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2.
Within the scope of the recombinant glycosyl transferase UGT72E3/2 protein of the present invention, a protein having an amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the protein are included. The term “functional equivalent” indicates a protein having, as a result of addition, substitution, or deletion of an amino acid, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 2, and it exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2. The term “substantially the same physiological activity” means an increase in syringin synthesis.
The present invention also includes a “fragment”, a “derivative”, and an “analogue” of the recombinant glycosyl transferase UGT72E3/2 protein. The terms “fragment”, “derivative”, and “analogue” that are used herein indicate a polypeptide having substantially the same biological function or activity as the recombinant glycosyl transferase UGT72E3/2 polypeptide of the present invention. The derivative and analogue of the present invention can be any one of the followings: (1) a polypeptide in which one or more conservative or non-conservative amino acid residue (preferably, a conservative amino acid residue) is substituted (the substituted amino acid may be either coded or not coded by genetic code), (2) a polypeptide having a substituent (s) on one or more amino acid, (3) a polypeptide derived from a mature polypeptide which is bonded to other compound (i.e., a compound capable of increasing the half life of a polypeptide; for example, polyethylene glycol), and (4) a polypeptide derived from aforementioned polypeptide which is bonded to an additional amino acid sequence (for example, a leading sequence, a secretory sequence, a sequence used for purification of the polypeptide, a proteinogen sequence or fusion protein). The fragment, derivative, and analogue defined herein are well known to a person skilled in the art.
The polynucleotide encoding the mature polypeptide represented by SEQ ID NO: 2 encompasses a coding sequence which encodes the mature polypeptide only; a sequence encoding the mature polypeptide and various additional coding sequences, and; a sequence encoding the mature polypeptide (and any additional coding sequence) and a non-coding sequence.
The term a “polynucleotide encoding a polypeptide” means a polynucleotide which encodes a polypeptide or a polynucleotide further comprising a coding and/or a non-coding sequence.
The present invention also relates to a polynucleotide variant which encodes the same amino acid sequence as the sequence described in the present invention, or a polypeptide containing its fragment, analogue, or derivative. The polynucleotide variant can be either a naturally occurring allele variant or a non-naturally occurring variant. The nucleotide variant encompasses a substitution variant, a deletion variant, and an insertion variant. As it is well known in the pertinent art, the allele variant is an alternative of a polynucleotide, and it may contain one or more substitution, deletion, or insertion of polynucleotide. However, no substantial functional change is yielded in the polynucleotide encoded by the variant.
The present invention also provides a gene encoding the recombinant glycosyl transferase UGT72E3/2 protein.
The gene encoding the recombinant glycosyl transferase UGT72E3/2 protein of the present invention is prepared from UGT72E3 and UGT72E2 genes originating from Arabidopsis thaliana by using a domain swapping method.
In the present invention, for having increased syringin production rate, the recombinant genes UGT72E2/3 and UGT72E3/2 were prepared from UGT72E2 and UGT72E3 genes by using a domain swapping method to produce a new recombinant glycosyl transferase having a high glycosyl transferase activity like UGT72E2 while maintaining the substrate specificity for sinapyl alcohol like UGT72E3.
First, the enzyme characteristics of the glycosyl transferase UGT72E clade which has been reported to have a capability of transferring a sugar to sinapyl alcohol, i.e., a precursor of syringin, or structurally similar coniferyl alcohol were determined for about 100 glycosyl transferases from Arabidopsis thaliana. The UGT72E clade also has structural characteristics that are similar to a common glycosyl transferase, and it has been reported that the amino terminal domain has a substrate recognition site, the carboxy terminal domain has an enzymatically activated region for transferring a sugar activated by UDP to a substrate, and the PSPG (Plant Secondary Product Glucosyl transferase) at the carboxyl terminal is particularly important for the activity of glycosyl transferase derived from a plant. The UGT72E clade includes glycosyl transferase UGT72E1, UGT72E2, and UGT72E3 having similar nucleotides.
In the present invention, each of the UGT72E2 and UGT72E3 was divided into an amino fragment including the amino acids from number 1 to number 344 and a carboxy fragment including the amino acids from number 345 to number 481. The amino fragment includes a region for determining the substrate recognition specificity and the carboxy terminal includes the PSPG motif, which is important for glycosyl transferase activity. For efficient production of syringin in a plant, the substrate specificity is more important than the glycosyl transferase activity. Thus, instead of having a precise half-cut, a large amino fragment is prepared to include 3⁄4 of the entire length while the carboxy fragment is prepared as a small fragment including PSPG motif. As a result, the recombinant UGT72E3/2 gene of the present invention was produced by linking an amino fragment including the amino acids from number 1 at the amino terminal to number 344 of UGT72E3 to a carboxy fragment including the amino acids from number 345 to number 481 at the carboxy terminal of UGT72E2.
The gene of the present invention can be either a DNA or an RNA which encodes the recombinant glycosyl transferase UGT72E3/2 protein. cDNA, genomic DNA, and an artificial synthetic DNA are included in DNA. DNA can be either single stranded or double-stranded. DNA can be either a coding sequence or a non-coding sequence.
Preferably, the gene of the present invention may contain the nucleotide sequence of SEQ ID NO: 1. Further, homologues of the nucleotide sequence are also within the scope of the present invention. Specifically, the above described gene may comprise a nucleotide sequence which has preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1. The “sequence homology %” for a certain polynucleotide is identified by comparing a comparative region with two sequences that are optimally aligned. In this regard, a part of the polynucleotide in comparative region may comprise an addition or a deletion (i.e., a gap) compared to a reference sequence (without any addition or deletion) relative to the optimized alignment of the two sequences.
The present invention also relates to a polynucleotide which hybridizes to a sequence having sequence homology of at least 50%, preferably at least 70%, and more preferably at least 80% with the nucleotide sequence of SEQ ID NO: 1 described above. The present invention particularly relates to a polynucleotide which hybridizes to the polynucleotide of the present invention under a stringent condition. As described herein, the “stringent condition” means (1) hybridization and washing at low ionic strength and high temperature like 0.2×SSC, 0.1% SDS, 60° C.; (2) hybridization in the presence of a denaturing agent like 50% (v/v) formamide, 0.1% bovine serum/0.1% Ficoll, and 42° C., or (3) hybridization occurring between only two sequences having homology of at least 80%, preferably at least 90%, and more preferably at least 95%. Further, the biological function and activity of the polypeptide encoded by a hybridizable nucleotide are the same as those of the mature polypeptide represented by SEQ ID NO: 2.
The present invention also provides a recombinant vector comprising the gene which encodes the aforementioned recombinant glycosyl transferase UGT72E3/2 protein.
The term “recombinant” indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, or a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene fragment in the form of a sense or antisense, which are not found in natural state of cell. In addition, a recombinant cell can express a gene that is found in natural state, provided that said gene is modified and re-introduced into the cell by an artificial means.
The recombinant plant expression vector of the present invention can be used as a transient expression vector which allows transient expression of a foreign gene in a plant and also as a plant expression vector which allows permanent expression of a foreign gene in a plant.
A binary vector which can be used for the present invention can be any binary vector comprising RB (right border) and LB (left border) of T-DNA which can transform a plant when it is present with Ti plasmid of A. tumefaciens. Preferably, pBI101 (Cat#: 6018-1, Clontech, USA), pBIN19 (Genbank Deposit No. U09365), pBI121, pCAMBIA and the like, which are often used by a skilled person in the pertinent art, are used.
The term “vector” is used herein to refer DNA fragment(s) and nucleotide molecules that are delivered to a cell. Vector can replicate DNA and be independently reproduced in a host cell. The terms “delivery system” and “vector” are often interchangeably used. The term “expression vector” means a recombinant DNA molecule comprising a desired coding sequence and other appropriate nucleotide sequences that are essential for the expression of the operably-linked coding sequence in a specific host organism. The promoter, enhancer, termination signal and polyadenylation signal which can be used in eukaryotic cells are well known in the art.
A preferred example of plant expression vector is Ti-plasmid vector which can transfer a part of itself, i.e., so called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vector (see, EP 0 116 718 B1) are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a genome of a plant. Especially preferred form of Ti-plasmid vector is a so-called binary vector which has been disclosed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other vector that can be used for introducing the gene of the present invention to a host plant can be selected from a double-stranded plant virus (e.g., CaMV), a single-stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector. Use of said vector can be advantageous especially when a plant host cannot be appropriately transformed.
Expression vector may comprise at least one selective marker. Said selective marker is a nucleotide sequence having a property that can make a target gene get selected by a common chemical method. Examples include all genes that are useful for distinguishing transformed cells from non-transformed cells. Specific examples include a gene resistant to herbicide such as glyphosate and phosphinotricine, and a gene resistant to antibiotics such as kanamycin, ampicillin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.
With regard to the plant expression vector according to one embodiment of the present invention, the promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS, and histone promoter, but not limited thereto. The term “promoter” means a DNA molecule to which RNA polymerase binds in order to initiate its transcription, and it corresponds to a DNA region upstream of a structural gene. The term “plant promoter” indicates a promoter which can initiate transcription in a plant cell. The term “constitutive promoter” indicates a promoter which is active in most of environmental conditions and development states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, the constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing the constitutive promoter is not limited herein.
In the above-described recombinant vector of the present invention, any kind of a typical terminator can be used. Examples include nopalin synthase (NOS), rice α-amylase RAmyl A terminator, phaseoline terminator, and a terminator for octopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto.
The present invention also provides a host cell transformed with the aforementioned recombinant vector.
When an eukaryotic cell is transformed with the vector of the present invention, yeast (Saccharomyces cerevisiae), an insect cell, a human cell (for example, CHO (Chinese hamster ovary) cell line, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell line), a plant cell, and the like can be used as a host cell. The host cell is preferably a plant cell.
As for the method to deliver the vector of the present invention to a holt cell, the vector can be introduced to a host cell by a microinjection method, calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, DEAE-dextran treatment method, or a gene bombardment method, and the like.
The present invention further provides a method of increasing syringin synthesis in a plant compared to the wild type, comprising transforming a plant cell with the recombinant vector to over-express the UGT72E3/2 gene.
According to the method of one embodiment of the present invention, the UGT72E3/2 gene may consist of the nucleotide sequence of SEQ ID NO: 1, but not limited thereto.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein, which consists of an amino acid sequence of SEQ ID NO: 2.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2 and a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of an amino acid sequence of SEQ ID NO: 4.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of an amino acid sequence of SEQ ID NO: 4, and a recombinant vector comprising the gene encoding the Myb58 protein which consists of an amino acid sequence of SEQ ID NO: 6.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of an amino acid sequence of SEQ ID NO: 4, and a recombinant vector comprising the gene encoding the Myb63 protein which consists of an amino acid sequence of SEQ ID NO: 8.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2 and a recombinant vector knock-outing the gene encoding the CHS (chalcone synthase) protein which consists of an amino acid sequence of SEQ ID NO: 10.
In the present invention, for efficient supply of sinapyl alcohol as a substrate for syringin to the transformant over-expressing the recombinant glycosyl transferase UGT72E3/2, each step and working enzymes of the synthesis pathway for syringin were utilized. Specifically, for enhancing the introduction of coumaroyl-CoA to the synthesis pathway for phenylpropanoid, HCT (hydroxycinamoyl-CoA: shikimate/quinqte hydroxycinamoyl transferase) gene was over-expressed and the amount of coniferyl aldehyde converted to coniferyl alcohol is reduced, and for promoting conversion to sinapyl alcohol, the F5H (ferulate 5-hydroxylase) gene was over-expressed. Further, in order to reduce the amount of coumaroyl-CoA lost from the synthesis pathway for syringin to the flavonoid pathway, a mutant deficient of the function of CHS (chalcone synthase) gene was used. For knock-out of the CHS gene, a silencing vector may be used, but it is not limited thereto.
The term “knock-out” as described herein means a modification or a removal of specific gene from a nucleotide sequence to prevent the expression of the specific gene, and it generally indicates a phenomenon in which expression of a gene is down-regulated or completely suppressed.
The present invention further provides a method for producing a transgenic plant with increased syringin synthesis compared to the wild type, comprising:
(a) transforming a plant cell with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2, and
(b) regenerating a plant from the transgenic plant cell of the step (a).
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4; and
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4;
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein;
(d) producing a transgenic plant over-expressing the Myb58 protein by transforming a plant with a recombinant vector comprising the gene encoding Myb58 protein which consists of an amino acid sequence of SEQ ID NO: 6; and
(e) cross-breeding the transgenic plant over-expressing simultaneously the UGT72E3/2 protein and F5H protein of the step (c) and the transgenic plant over-expressing the Myb 58protein of the step (d) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein, F5H protein, and Myb58 protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4;
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein;
(d) producing a transgenic plant over-expressing the Myb63 protein by transforming a plant with a recombinant vector comprising the gene encoding Myb63 protein which consists of an amino acid sequence of SEQ ID NO: 8; and
(e) cross-breeding the transgenic plant over-expressing simultaneously the UGT72E3/2 protein and F5H protein of the step (c) and the transgenic plant over-expressing the Myb63 protein of the step (d) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein, F5H protein, and Myb63 protein.
The present invention further provides a method for producing a transgenic plant with increased syringin production compared to the wild type, comprising
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene encoding the recombinant glycosyl transferase UGT72E3/2 protein which consists of an amino acid sequence of SEQ ID NO: 2;
(b) producing a plant in which the gene encoding the CHS (chalcone synthase) protein consisting of an amino acid sequence of SEQ ID NO: 10 is knocked out; and
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the plant with knocked-out CHS protein-coding gene of the step (b) and selecting a transgenic plant which over-expresses the UGT72E3/2 protein and suppresses expression of the CHS protein.
With the method according to one embodiment of the present invention, the plant exhibits increased syringin synthesis preferably in the leaves or roots, or most preferably in the leaves, but it is not limited thereto.
The method of the present invention comprises a step of transforming a plant cell with the recombinant vector of the present invention, and the transformation may be mediated by Agrobacterium tumefaciens. Further, the method of the present invention comprises a step of regenerating a transgenic plant from the transgenic plant cell. As for the method for regenerating a transgenic plant from a transgenic plant cell, a method well known in the pertinent art can be used.
The transgenic plant cell needs to be regenerated into a whole plant. Techniques for regeneration into a mature plant by culture of callus or protoplast are well known in the pertinent art for various 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 for positive regulation of the genes that are involved with the biosynthesis pathway of phenylpropanoid, and Myb63 known to have a similar function is also expected to exhibit the synergistic effect in the process of syringin synthesis.
The present invention further provides a transgenic plant with increased syringin production compared to the wild type, which is produced by each method described above, and a seed thereof.
With regard to the plant according to one embodiment of the present invention, the plant can be preferably a dicot plant such as Arabidopsis thaliana, tobacco, eggplant, pepper, tomato, burdock, crown daisy, lettuce, balloon flower, spinach, chard, yam, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, Raphanus sativus for. raphnistroides MAK, watermelon, oriental melon, cucumber, zucchini, gourd, strawberry, soybean, mung bean, kidney bean, or sweet pea. Most preferably, it can be Arabidopsis thaliana, but it is not limited thereto.
The present invention still further provides a composition for increasing syringin synthesis in a plant comprising, as an effective component, a recombinant vector comprising the gene consisting of SEQ ID NO: 1 which encodes the UGT72E3/2 protein. The composition comprises, as an effective component, the recombinant vector comprising the gene consisting of SEQ ID NO: 1 which encodes the UGT72E3/2 protein, and according to transformation of a plant with the recombinant vector, it is possible to increase the syringin synthesis in plant.
Herein below, the present invention is explained in greater detail in view of the Examples. However, it is evident that the following Examples are given only for exemplification of the present invention and by no means the present invention is limited to the following Examples.
Information of Genes and Nucleotide Sequences used in the Present Invention
Information of the genes and nucleotide sequences that are used in the present invention is as described in the following Table 1.











TABLE 1








Nucleotide sequence
Amino acid sequence



Gene name
number
number




















UGT72E3/2
1
2



F5H
3
4



Myb58
5
6



Myb63
7
8



CHS
9
10



HCT
11
12










EXAMPLE 1
Primary and Secondary Structures of Arabidopsis Thaliana Glycosyl Transferase UGT72E2 and UGT72E3
In order to develop a new glycosyl transferase which is useful for in-plant production of syringin having various pharmaceutical applications, a comparison of primary and secondary structures is made between UGT72E2 having very excellent glycosyl transferase efficiency but poor substrate specificity for coniferyl alcohol, which is a precursor of syringin, and UGT72E3 protein having very excellent substrate specificity for sinapyl alcohol but poor glycosyl transferase efficiency, by using SWISS-MODEL workspace (FIG. 1). As a pre-step for the domain swapping to produce a new recombinant glycosyl transferase having all the advantages of both enzymes, a difference in the primary and secondary structures of the carboxy terminal containing PSPG motif that is important for glycosyl transferase activity and the amino terminal for determining the substrate specificity and the region in which domain swapping occurs were marked.
EXAMPLE 2
Tertiary Structure of Arabidopsis Thaliana Glycosyl Transferase UGT72E2 and UGT72E3, and Newly Produced Recombinant Glycosyl Transferase UGT72E2/3 and UGT72E3/2
As it is shown from glycosyl transferase UGT72B1 and VvGT1 derived from Arabidopsis thaliana and grape, respectively, of which tertiary structure has been already studied thoroughly, a glycosyl transferase generally has a region for determining substrate specificity (i.e., sugar acceptor) and a region for determining glycosyl transferase activity (i.e., sugar donor), which are adjacent to each other in a deep and narrow gap at a boundary between the amino terminal domain and carboxy terminal domain (FIGS. 2A and 2B). By using SWISS-MODEL workspace, the expected tertiary structure of UGT72E2, UGT72E3, UGT72E2/3 and UGT72E3/2 was obtained (FIG. 2C). It is believed that a structural change near the region for determining the substrate specificity and the region for determining the glycosyl transferase activity is involved with a difference in the substrate specificity and glycosyl transferase activity among glycosyl transferases of UGT72E2, UGT72E3, UGT72E2/3 and UGT72E3/2.
EXAMPLE 3
Recombinant Vector used for Transformant Production, Expression Level of Gene Transferred to Transformant, and Phenotype of Transformant in Response to UV Rays
Inventors of the present invention investigated, among one hundred glycosyl transferases of Arabidopsis thaliana, the enzyme characteristics of the glycosyl transferase UGT72E clade which has been reported to have an ability of transferring a sugar to sinapyl alcohol as a precursor of syringin or structurally similar coniferyl alcohol. UGT72E clade also has structural characteristics that are similar to those of a common glycosyl transferase. The amino terminal domain has a region for substrate recognition and the carboxy terminal has an enzyme activated region for transferring a sugar activated by UDP to a substrate. In particular, PSPG (Plant Secondary Product Glucosyl transferase) motif at the carboxy terminal is reported to be important for the activity of a glycosyl transferase derived from a plant. The UGT72E clade includes glycosyl transferase UGT72E1, UGT72E2 and UGT72E3 having similar nucleotide sequence.
In the present invention, each of the UGT72E2 and UGT72E3 was divided into an amino fragment including the amino acids from number 1 to number 344 and a carboxy fragment including the amino acids from number 345 to number 481. The amino fragment includes a region for determining the substrate recognition specificity and the carboxy terminal includes the PSPG motif, which is important for glycosyl transferase activity. For efficient production of syringin in a plant, the substrate specificity is more important than the glycosyl transferase activity. Thus, instead of having a precise half-cut, a large amino fragment is prepared to include 3⁄4 of the entire length while the carboxy fragment is prepared to as a small fragment including PSPG motif.
The recombinant UGT72E2/3 gene used in the present invention was prepared by linking an amino fragment including the amino acids from number 1 at the amino terminal to number 344 of UGT72E2 to a carboxy fragment including the amino acids from number 345 to number 481 at the carboxy terminal of UGT72E3. The recombinant UGT72E3/2 gene was prepared by linking an amino fragment including the amino acids from number 1 at the amino terminal to number 344 of UGT72E3 to a carboxy fragment including the amino acids from number 345 to number 481 at the carboxy terminal of UGT72E2.
In the present invention, a binary vector was constructed such that the coding regions of UGT72E2 and UGT72E3 genes isolated from Arabidopsis thaliana and the recombinant UGT72E2/3 and UGT72E3/2 genes, which have been newly produced in the present invention, are controlled by CaMV35S promoter and a super promoter. The resulting vector was incorporated into A. tumefaciens EHA105, and then Arabidopsis thaliana was transformed with the bacteria using an in planta method (FIG. 3).
Because the phenotype of a transformant is significantly affected by expression level of an introduced transfer gene, among the Arabidopsis thaliana transformants with hygromycin resistance, the expression amount resulting from stable introduction of the transferase genes UGT72E2, UGT72E3, UGT72E2/3 and UGT72E3/2 derived from Arabidopsis thaliana was determined based on RT-PCR. For specific amplification of the introduced gene, a combination of UGT72E gene-specific forward primer (5′GGTTGGAGCTCGACGTTGGAAAGCGTC 3′; SEQ ID NO: 13) and a reverse primer (5′TTAAAGCAGGGCATGCCTGC 3′; SEQ ID NO: 14) specific to 3′ UTR region of the vector was used. For correction of relative RNA amount, Actin 1 gene from Arabidopsis thaliana, which is always expressed at constant level, was used as a reference gene. Among fifteen transformants for each gene which has been determined, transformant lines having similar expression of UGT72E2, UGT72E3, UGT72E2/3 and UGT72E3/2 were finally identified (FIG. 3B).
Conversion of sinapyl alcohol to syringin according to activation of a glycosyl transferase in leaves reduce the production of sinapyl ester which absorbs UV rays in leaves. As such, when the leaves of a transformant with enhanced glycosyl transferase activity is exposed to UV rays, UV rays are mostly absorbed by chlorophylls to exhibit a very strong red color. When production of sinapyl ester occurs normally in leaves, the UV ray absorption amount by chlorophylls is reduced to exhibit a very weak red color (FIG. 3C).
EXAMPLE 4
Quantitative HPLC Analysis of Coniferin and Syringin Production in Leaves of Transformant
Monolignol produced by activation of synthesis pathway for phenylpropanoid in leaves of an angiosperm plant is mostly present as coniferyl alcohol, which is converted to coniferin by a glycosyl transferase. With an enzyme action, an extremely small portion of coniferyl alcohol is converted to sinapyl alcohol as a precursor of syringin, and rate of conversion to syringin caused by glycosyl transferase is very low due to low enzyme activity. Most of the sinapyl alcohol is converted to sinapyl ester which has an activity of protecting leaves by absorbing UV rays. As such, the over-expression of UGT72E2 gene having high substrate specificity for coniferyl alcohol increases coniferin production and over-expression of UGT72E3 having high substrate specificity for sinapyl alcohol relatively increases production of syringin (FIGS. 4A and 4B). Over-expression of the recombinant gene UGT72E3/2 exhibited the effect of increasing syringin production in plant by 48.7% compared to the over-expression of the UGT72E3 gene (FIGS. 4B and 4C).
EXAMPLE 5
Quantitative HPLC Analysis of Coniferin and Syringin Production in Roots of Transformant
Unlike the leaves that are constantly exposed to light, once exposed to light, the roots of a plant show increased expression of various genes that are involved with the synthesis pathway for phenylpropanoid based on a signal transduction mechanism. As a result, synthesis of monolignols including a great amount of coniferyl alcohol and sinapyl alcohol is enhanced. Although the production of coniferin and syringin that have been hardly detected in leaves of the wild type plant cultivated under same condition was significantly increased, the production rate of syringin was still low, i.e., only 25% of the coniferin. Further, although the production of coniferin was increased by two or more times in the roots of a transformant which over-expresses UGT72E2 having high glycosyl transferase activity and strong substrate specificity for coniferyl alcohol, there was no big change in syringin production (FIGS. 5B and 5C). In the roots of a transformant which over-expresses UGT72E3 gene having weak glycosyl transferase activity but strong substrate specificity for sinapyl alcohol, coniferin and syringin were produced at almost the same level as those of the wild type. However, the over-expression of the recombinant gene UGT72E3/2 exhibited the effect of increasing syringin production in the roots by 11.7% compared to the over-expression of the UGT72E3 gene (FIGS. 5B and 5C).
EXAMPLE 6
Nucleotide Sequence and Amino Acid Sequence of Glycosyl Transferase UGT72E3/2
Nucleotide sequence of the new recombinant glycosyl transferase UGT72E3/2 gene and the amino acid sequence of the protein encoded by the nucleotide sequence, which exhibits the glycosyl transferase activity enhanced by 48.7% or more compared to the conventionally known glycosyl transferase UGT72E3 while maintaining the substrate specificity for sinapyl alcohol, are shown in FIG. 6.
EXAMPLE 7
Comparison of Activity of Glycosyl Transferases that are Present in Protein Extract Prepared from Leaves of Wild Type and Transgenic Arabidopsis Thaliana Over-Expressing Each of the Glycosyl Transferase UGT72E2, UGT72E3, UGT72E2/3, and UGT72E3/2
Most of monolignols produced by activation of synthesis pathway for phenylpropanoid in leaves of an angiosperm plant are present as coniferyl alcohol, and it is converted to coniferin while only an extremely small portion is converted to sinapyl alcohol as a precursor of syringin by continuous enzyme action by F5H (furulate 5-hydroxylase), COMT (caffeic acid 3-O-methyltransferase) and CAD. As such, supply of sinapyl alcohol as a substrate is an important factor for determining syringin production together with the activity of glycosyl transferase.
In order to compare the activity of the glycosyl transferases, 1 mM coniferyl alcohol or sinapyl alcohol and 5 mM UDP-glucose were added as a substrate to a protein extract prepared from leaves of wild type and transgenic Arabidopsis thaliana over-expressing each of the glycosyl transferase UGT72E2, UGT72E3, UGT72E2/3, and UGT72E3/2. Then, the reaction was allowed to occur at 22° C. for 60 minutes. After that, methanol (2× volume) was added to the reaction solution to terminate the reaction, and by using HPLC, coniferin and syringin produced before and after the reaction were quantified by HPLC. As it has been previously shown under in vivo conditions, the recombinant glycosyl transferase UGT72E3/2 has high substrate specificity for sinapyl alcohol and high glycosyl transferase activity. In particular, UGT72E3/2 exhibited better syringin production speed according to addition of substrate than other glycosyl transferases (FIG. 7).
The above results demonstrate that, by also using a method of increasing supply of sinapyl alcohol based on a metabolic engineering means to a transformant over-expressing UGT72E3/2, syringin can be produced in a plant at high efficiency.
EXAMPLE 8
Synthesis Pathway for Phenylpropanoid and Gene Regulation Site for Syringin Synthesis
For efficient supply of sinapyl alcohol as a substrate for syringin to a transformant over-expressing the recombinant glycosyl transferase UGT72E3/2 with use of a metabolic engineering means, each step and working enzymes of the synthesis pathway for phenylpropanoid were utilized. Specifically, in order to reduce the amount of coumaroyl-CoA lost from the synthesis pathway for syringin to the flavonoid pathway, a mutant deficient of CHS (chalcone synthase) gene was used. Further, for enhancing the introduction of coumaroyl-CoA to the synthesis pathway for phenylpropanoid, HCT (hydroxycinamoyl-CoA: shikimate/quinqte hydroxycinamoyl transferase) gene was over-expressed, and the amount of coniferyl aldehyde converted to coniferyl alcohol is reduced, and for promoting conversion to sinapyl alcohol, a strategy of over-expressing the F5H (ferulate 5-hydroxylase) gene was employed (FIG. 8).
EXAMPLE 9
Production of Transgenic Arabidopsis Thaliana Over-Expressing HCT, F5H and Myb58 as a Gene for Regulating Synthesis Pathway for Phenylpropanoid and Determination of Expression Amount of Each Gene
A binary vector was constructed such that the coding regions of HCT, F5H and Myb58 of Arabidopsis thaliana are regulated by a super promoter. After introducing the vector to Agrobacterium tumefaciens EHA105, transformation of Arabidopsis thaliana was performed by in planta method using the aforementioned bacteria. At that time, for the purpose of having pyramidal accumulation of HCT, F5H and Myb58 genes in the UGT72E3/2 over-expressing transformant, which has been produced by using hygromycin resistant selection marker, by a cross-breeding and selection mode, the Arabidopsis thaliana transformant of each gene was prepared by using kanamycin (HCT and F5H) or herbicide (Myb58) resistant selection marker.
Because the phenotype of a transformant is significantly affected by expression level of an introduced transfer gene, among the transformants which have been selected, the expression amount resulting from stable introduction of HCT, F5H and Myb58 genes was determined based on RT-PCR. For specific amplification of the introduced gene, a combination of HCT gene-specific forward primer HCT-F (5′-CTGGTTACTTTGGGAATGTGATATTCAC-3; SEQ ID NO: 15), F5H gene-specific forward primer a F5H-F (5′-CAGACGAGTTGAAGAATCCGACATCGAG-3; SEQ ID NO: 16), and Myb58 gene-specific forward primer Myb58-F (5′-CAGACGAGTTGAAGAATCCGACATCGAG-3; SEQ ID NO: 17) were used. Further, as a reverse primer, UTR-R (5′TTAAAGCAGGGCATGCCTGC 3′; SEQ ID NO: 14) specific to 3′ UTR region of the vector was used. For calibration of the relative amount of RNA, Actin 2 gene of Arabidopsis thaliana, which is always expressed at constant level, was used as a reference gene. Ten transformants were examined for each gene, and as a result, transformant lines having excellent expression of HCT, F5H and Myb58 were finally obtained (FIG. 9). Meanwhile, there is only one CHS gene present Arabidopsis thaliana, and when this gene is deficient, color of the seed coat turns into yellow color. Based on such difference in the phenotype, the homozygous transformant was isolated.
EXAMPLE 10
Quantitative HPLC Analysis of Coniferin and Syringin Production in Leaves and Roots of Transformant
Via the synthesis pathway for phenylpropanoid, an angiosperm plant produces the following three kinds of monolignol; i.e., H monolignol using p-coumaryl alcohol, G monolignol using coniferyl alcohol, and S monolignol using sinapyl alcohol. However, most monolignols are present in G monolignol form, and thus the concentration of coniferyl alcohol is relatively the highest in a plant cell. Over expression of the glycosyl transferase UGT72E2 yields conversion of coniferyl alcohol at high concentration to coniferin. Because part of coniferyl alcohol is converted to sinapyl alcohol due to the enzyme action of F5H and COMT (caffeic acid 3-O-methyltransferase), concentration of sinapyl alcohol as a precursor of syringin is very low in a plant cell. Thus, for efficient production of syringin in a plant cell, a metabolic engineering approach for increasing sinapyl alcohol in addition to having glycosyl transferase UGT72E3/2 with high efficiency is needed.
In order to examine a synergistic effect between the glycosyl transferase UGT72E3/2 gene and the HCT, F5H and CHS genes, quantitative HPLC analysis of coniferin and syringin production in leaves and roots of the transformant was performed. Specifically, a transformant over-expressing HCT or F5H, which regulates an important step of the synthesis pathway for phenylpropanoid, was produced and subjected to cross-breeding with the transformant over-expressing the glycosyl transferase UGT72E3/2 with high efficiency. After that, a transformant over-expressing HCT and UGT72E3/2 or F5H and UGT72E3/2 was separated at F2 generation, and the homozygous line was obtained from the next generation. Further, for preventing the loss of p-coumaroyl-CoA as an important precursor of the synthesis pathway for phenylpropanoid to the synthesis pathway for flavonoid, a mutant deficient of CHS (chalcone synthase), which is responsible for conversion of p-coumaroyl-CoA to chalcone, and the transformant over-expressing UGT72E3/2 were subjected to cross-breeding. After isolating the line with knocked-out CHS gene and over-expressed UGT72E3/2 gene from F2 generation, the homozygous line was confirmed from the next generation.
As a result of determining the efficiency of syringin synthesis in leave and roots of the transformant by using HPLC, it was found that, compared to the transformant in which only the UGT72E3/2 gene is over-expressed, the syringin synthesis was increased by 17.3%, 71.3% and 64.6%, respectively, in the leaves of the plant with over-expressed HCT or over-expressed F5H, but with deficient CHS gene function (FIG. 10).
However, from the roots of the plant, only the transformant line having over-expression of UGT72E3/2 and F5H showed slightly increased syringin synthesis compared to the transformant in which only the UGT72E3/2 gene is over-expressed (FIG. 11).
EXAMPLE 11
Quantitative HPLC Analysis of Syringin Production in Leaves and Roots of Transformant Based on Synergistic Effect using Pyramiding of UGT72E3/2, F5H and Myb58 Genes
Accumulated over-expression of the new glycosyl transferase gene UGT72E3/2 having high specificity for sinapyl alcohol, which has been developed by an enzyme engineering method, and F5H gene involved with conversion of coniferyl alcohol to sinapyl alcohol in the phenylpropanoid pathway exhibited the effect of remarkably increasing the syringin production rate. Meanwhile, when exposed to light, roots of a plant exhibit increased expression of various genes that are involved with the synthesis pathway of phenylpropanoid based on a light signal transduction mechanism. As a result, synthesis of monolignols including a large amount of coniferyl alcohol and sinapyl alcohol is enhanced, and thus the synthesis amount of syringin in roots of the wild type plant is at least 36 times higher than that of the leaves. Meanwhile, the production amount of syringin in leaves of the transformant which over-expresses UGT72E3/2 and F5H is increased by 16 times or more than that of the leaves of the wild type. However, compared to the syringin amount produced in the roots of the transformant, it is still lower by a factor of at least 4. Increasing the syringin production in leaves has an advantage that, unlike roots, a large scale production can be made without destroying a plant. As such, like the roots exposed to light, a method of increasing various genes involved with the phenylpropanoid synthesis is also needed for leaves.
In order to solve the aforementioned problems, syringin production by a transformant based on synergistic effect using pyramiding of the glycosyl transferase UGT72E3/2 gene, F5H gene of the synthesis pathway for phenylpropanoid, and Myb58 gene as a transcription factor for positive regulation of the genes relating to the synthesis pathway of lignin was measured in the present invention by quantitative HPLC analysis.
To confirm the synergistic effect using the Myb58 gene, a transformant over-expressing the Myb58 gene as a specific transcription factor for positive regulation of the synthesis pathway of lignin in Arabidopsis thaliana was produced. Since the Myb58 gene cannot enhance the expression of F5H gene, after cross-breeding with a transformant which over-expresses both UGT72E3/2 and F5H genes, a transformant line which over-expresses all of the Myb58, UGT72E3/2 and F5H genes was selected at F2 generation and the homozygous line was obtained at the next generation. As a result of investigating the efficiency for syringin synthesis in the leaves and roots of those transformants by using HPLC, it was found that, in the leaves of the transformant in which all of the UGT72E3/2, F5H, and Myb58 genes are over expressed, the production amount of syringin is increased by 8 times compared to the transformant in which UGT72E3/2 and F5H genes are over-expressed, or by 10 times compared to the transformant in which only UGT72E3/2 gene is over-expressed, thus indicating an excellent effect (FIG. 12).
The results of having efficient production of syringin in the leaves based on the synergistic effect among three genes that are related to syringin production, i.e., UGT72E3/2, F5H, and Myb58 genes, are in match with the results of the analysis conducted at gene level. By enhancing the expression of various genes relating to the synthesis pathway of phenylpropanoid by over-expression of Myb58 and separately over-expressing UGT72E3/2 and F5H genes that are not regulated by Myb58, the syringin production amount is remarkably increased in the leaves of the transformant (FIG. 14). Further, it was shown that the syringin production amount in the leaves of the transformant is increased by 2 times or more compared to the production amount in the roots. Thus, it was confirmed that, as a synergistic effect caused by over-expression of UGT72E3/2, F5H and Myb58 genes, the syringin production is most ideally reduced by 2 times or so in the roots while it is significantly increased in the leaves. Accordingly, a transgenic plant capable of producing syringin in a large amount was established with the aforementioned method.

## Claims

1. Recombinant glycosyl transferase UGT72E3/2 protein consisting of the amino acid sequence of SEQ ID NO: 2.

2. A gene encoding the UGT72E3/2 protein of claim 1.

3. A recombinant vector comprising the gene encoding the UGT72E3/2 protein of claim 2.

4. A host cell transformed with the recombinant vector of claim 3.

5. The host cell according to claim 4, characterized in that it is a plant cell.

6. A method of increasing syringin synthesis in a plant compared to the wild type, comprising transforming a plant cell with the recombinant vector of claim 3 to over-express the UGT72E3/2 gene.

7. A transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene of claim 2.

8. A transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene of claim 2 and a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of the amino acid sequence of SEQ ID NO: 4.

9. A transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene of claim 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of the amino acid sequence of SEQ ID NO: 4, and a recombinant vector comprising the gene encoding the Myb58 protein which consists of the amino acid sequence of SEQ ID NO: 6.

10. A transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene of claim 2, a recombinant vector comprising the gene encoding the F5H (furulate 5-hydroxylase) protein which consists of the amino acid sequence of SEQ ID NO: 4, and a recombinant vector comprising the gene encoding the Myb63 protein which consists of the amino acid sequence of SEQ ID NO: 8.

11. A transgenic plant with increased syringin production compared to the wild type, in which the plant is transformed with a recombinant vector comprising the gene of claim 2 and a recombinant vector comprising the gene encoding the CHS (chalcone synthase) protein which consists of the amino acid sequence of SEQ ID NO: 10.

12. A method for producing a transgenic plant with increased syringin synthesis compared to the wild type, comprising:
(a) transforming a plant cell with a recombinant vector comprising the gene of claim 2; and
(b) regenerating a plant from the transgenic plant cell of the step (a).

13. A method for producing a transgenic plant with increased syringin production compared to the wild type, comprising:
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene of claim 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4; and
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein.

14. A method for producing a transgenic plant with increased syringin production compared to the wild type, comprising:
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene of claim 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4;
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein;
(d) producing a transgenic plant over-expressing the Myb58 protein by transforming a plant with a recombinant vector comprising the gene encoding Myb58 protein which consists of an amino acid sequence of SEQ ID NO: 6; and
(e) cross-breeding the transgenic plant over-expressing simultaneously the UGT72E3/2 protein and F5H protein of the step (c) and the transgenic plant over-expressing the Myb 58 protein of the step (d) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein, F5H protein, and Myb58 protein.

15. A method for producing a transgenic plant with increased syringin production compared to the wild type, comprising:
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene of claim 2;
(b) producing a transgenic plant over-expressing the F5H (furulate 5-hydroxylase) protein by transforming a plant with a recombinant vector comprising the gene encoding F5H protein which consists of an amino acid sequence of SEQ ID NO: 4;
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the transgenic plant over-expressing the F5H protein of the step (b) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein and F5H protein;
(d) producing a transgenic plant over-expressing the Myb63 protein by transforming a plant with a recombinant vector comprising the gene encoding Myb63 protein which consists of an amino acid sequence of SEQ ID NO: 8; and
(e) cross-breeding the transgenic plant over-expressing simultaneously the UGT72E3/2 protein and F5H protein of the step (c) and the transgenic plant over-expressing the Myb63 protein of the step (d) and selecting a transgenic plant which over-expresses simultaneously the UGT72E3/2 protein, F5H protein, and Myb63 protein.

16. A method for producing a transgenic plant with increased syringin production compared to the wild type, comprising:
(a) producing a transgenic plant over-expressing the UGT72E3/2 protein by transforming a plant with a recombinant vector comprising the gene of claim 2;
(b) producing a plant in which the gene encoding the CHS (chalcone synthase) protein consisting of an amino acid sequence of SEQ ID NO: 10 is knocked out; and
(c) cross-breeding the transgenic plant over-expressing the UGT72E3/2 protein of the step (a) and the plant with knocked-out CHS protein-coding gene of the step (b) and selecting a transgenic plant which over-expresses the UGT72E3/2 protein and suppresses expression of the CHS protein.

17. A transgenic plant with increased syringin production compared to the wild type, which is produced by the method described in claim 12.

18. The plant according to claim 17, in which the plant is a dicot plant.

19. A seed of the transgenic plant of claim 17, wherein the seed comprises the recombinant vector.

20. A composition for increasing syringin synthesis in a plant comprising, a recombinant vector comprising the gene consisting of the nucleotide sequence of SEQ ID NO: 1 which encodes the UGT72E3/2 protein.