KR102053089B1 - Gene encoding recombinant UGTase protein increasing coniferin synthesis in plant and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant glycosyl transferase UGT72E2/3-1-myc or UGT72E2/3-2-myc protein based on an amino acid sequence of a sequence number 2 or a sequence number 4 capable of increasing coniferin synthesis in plants, and a use thereof. The recombinant glycosyl transferase of the present invention is expected to develop a high value agricultural biological material industry.

Description

Gene encoding recombinant UGTase protein increasing coniferin synthesis in plant and uses according to the present invention.

The present invention relates to recombinant sugar transfer enzymes UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc protein coding genes and their use to increase coniferin synthesis in plants.

Coniferin is a lignin-based glycoside, which is produced by the glycoside of the lignin-containing coniferyl alcohol (G type monolignol), which is produced through the synthesis path of phenylpropanoid, by glycotransferase (UDP-glucosyltransferase). do. In the Arabidopsis, a model plant, there are about 100 types of glycotransferase enzymes, and glycosides of various compounds including plant hormones are reported to be involved in inactivation of cells and storage in vacuoles. Among these, the sugar transfer enzyme UGT72E2 (UDP-glycosyltransferase 72E2) has a relatively high enzyme activity but low substrate specificity for coniferyl alcohol and sinapyl alcohol, depending on the concentration of the substrate. There is a drawback of synthesizing by mixing. Therefore, the development of a new glycotransferase with enhanced substrate specificity that can effectively produce coniferin in plants is a challenge that must be solved before mass production and application of the functional secondary metabolite, coniferin.

Coniferin is a representative pharmacological ingredient that is produced in the roots and stems of Ogapi and thorn ogapi, and it inhibits the activity of collagenase, collagenase, and elastase, an enzyme of elastin. It is excellent and has been reported to have excellent skin whitening effect by inhibiting melanin synthesis. Therefore, coniferin can be widely used as a material in various fields such as pharmaceutical industry, food industry and cosmetics industry.

On the other hand, Korean Patent No. 1399944 discloses a method for producing a transgenic soybean plant with increased contents of sirin and coniferin and a soybean plant using a gene encoding a recombinant transfer enzyme UGT72E-3 / 2 protein. In addition, Korean Patent Publication No. 2018-0060718 discloses a 'cosmetic composition comprising coniferin', but the present invention using a recombinant glycotransferase enzyme UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc protein coding gene There is no description of recombinant translocation enzyme protein coding genes and their use to increase the synthesis of coniferin in the plants of the invention.

The present invention is derived from the above requirements, the inventors of the present invention, the sugar transfer enzymes UGT72E2 and UGT72E3 (UDP- Recombinant genes UGT72E2 / 3-1 and UGT72E2 / 3-2 were prepared from the gene encoding glycosyltransferase 72E3) using a domain swapping method and 42 Myc epitopes were included at the carboxy terminus of the recombinant protein. New recombinant sugar transfer enzymes UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc with an amino acid sequence were prepared. In order to investigate the enzymatic properties of the recombinant genes, Arabidopsis transformants overexpressing UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc genes were prepared using Agrobacterium. As a result of quantitative comparison of the synthetic efficiency, it was confirmed that the transformants overexpressing the newly produced recombinant glycotransferases UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc significantly increased coniferin synthesis compared to wild type. In addition, UGT72E2-myc showed high activity against both cinafil alcohol and coniferyl alcohol in the in vitro reaction in which high concentration of substrate was added to the whole protein isolated from the leaves of the transformants, while UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc showed high activity only for coniferyl alcohol. The new glycotransferases UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc enhance both enzyme activity and substrate specificity compared to wild type. To confirm that Written, it has completed the present invention.

In order to solve the above problems, the present invention provides a recombinant sugar transfer enzyme UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

The present invention also provides a gene encoding the UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein.

The present invention also provides a recombinant vector comprising the gene.

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

In addition, the present invention comprises transforming plant cells with the recombinant vector, and overexpressing a gene encoding a UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein. coniferin) provides a method for increasing synthesis.

In addition, the present invention provides a method for producing a transformed plant with increased coniferin synthesis compared to the wild type comprising the step of transforming plant cells with the recombinant vector: and regenerating the plant from the transformed plant cells. to provide.

In addition, the present invention provides a transformed plant and its transformed seed having increased coniferin synthesis compared to the wild type produced by the production method.

In addition, the present invention provides a gene encoding the recombinant glycotransferase UGT72E2 / 3-1-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or the recombinant glycotransferase UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 4 It provides a composition for enhancing the synthesis of coniferin in a plant containing a recombinant vector comprising as an active ingredient.

The present invention is a recombinant sugar transfer enzyme UGT72E2 / 3 that can effectively mass-produce plant-derived coniferin from plants, which can be widely used as a material in various fields such as pharmaceuticals, foods and cosmetics industry because of excellent skin wrinkle improvement and whitening effect. Providing coding genes of -1-myc and UGT72E2 / 3-2-myc proteins is expected to enable the development of high value-added agricultural biological material industries.

1 is a result of measuring the amount of coniferin and sirin produced in the leaves of the transformant overexpressing the Arabidopsis wild-type and UGT72E2 or UGT72E2 / 3-2, respectively, by HPLC analysis.
FIG. 2 shows the results of in vitro reaction of coniferin alcohol or cinafil alcohol added to the whole protein isolated from the leaves of the transformant overexpressing the Arabidopsis wild-type and UGT72E2 or UGT72E2 / 3-2, respectively. Coniferin and Sirin production were quantitatively shown by HPLC analysis. 0 min: concentration of metabolite present in total protein extract added before reaction, 30 min: sum of concentration of metabolite present in total protein extract added and concentration of metabolite generated after 30 min reaction.
Fig. 3 shows the formation of coniferin and sirine in the leaves of a transformant overexpressing the Arabidopsis wild-type and three sugar transferases of UGT72E2-myc, UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc, respectively. HPLC chromatograms for quantitative analysis are shown. Peak 1 represents Coniferyl Alcohol 4-O-Glucoside (Coniferin) and Peak 2 represents Cinafil Alcohol 4-O-Glucoside (Cyrazine).
Figure 4 shows the HPLC and amount of coniferin and sirine production in the leaves of transformants overexpressing the Arabidopsis wild-type and three sugar transfer enzymes of UGT72E2-myc, UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc, respectively. It is shown quantitatively through analysis.
5 shows conies added to the whole protein isolated from the leaves of the transformant overexpressing the Arabidopsis wild-type and three types of sugar transfer enzymes, UGT72E2-myc, UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc, respectively. The amount of coniferin and sirin produced as a result of in vitro reaction of perrin alcohol or cinafil alcohol is quantitatively expressed through HPLC analysis. 0 min: concentration of metabolite present in total protein extract added before reaction, 30 min: sum of concentration of metabolite present in total protein extract added and concentration of metabolite generated after 30 min reaction.

In order to achieve the object of the present invention, the present invention provides a recombinant sugar transfer enzyme UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

The range of recombinant sugar transfer enzymes UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc proteins according to the present invention includes proteins having amino acid sequences represented by SEQ ID NOs: 2 and 4 and functional equivalents of the proteins, respectively. do. By "functional equivalent" is at least 70%, preferably at least 80%, more preferably at least 90%, even more than the amino acid sequence represented by SEQ ID NOs: 2 and 4 as a result of the addition, substitution or deletion of amino acids Preferably it refers to a protein having a sequence homology of 95% or more, and exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2 or SEQ ID NO: 4. "Substantially homogeneous physiological activity" means an increase in coniferin synthesis in a plant.

In the present invention, UGT72E2 and UGT72E3 proteins are used to prepare a novel recombinant glycotransferase that enhances the sugar transfer activity and substrate specificity for coniferin alcohol than UGT72E2 to increase the specific production rate of coniferin in plants. Recombinant genes UGT72E2 / 3 and UGT72E3 / 2 were prepared using domain swapping methods from the coding genes, and 42 amino acid sequences comprising three myc epitopes at the carboxy terminus of the protein expressed from the recombinant gene. The new recombinant glycotransferases UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc were added.

The present invention also provides a gene encoding the recombinant sugar transfer enzyme UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein.

The gene encoding the recombinant sugar transfer enzymes UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc protein according to the present invention may be composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The base sequence of the present invention was prepared by using a method of domain swapping from UGT72E3 and UGT72E2 genes derived from Arabidopsis and the addition of a myc epitope coding sequence to the carboxy terminus.

In the present invention, UGT72E2 (protein GenBank accession no .: AAL47362.1, gene GenBank accession no .: AY064651.1) and UGT72E3 (protein GenBank accession no .: ABO38789.1, gene GenBank accession no .: BT030376.1), respectively The amino fragments were divided into amino fragments comprising amino acids 1 to 344 or amino acids 1 to 240 at the amino terminus and carboxy fragments containing amino acids 345 or 481 to the carboxy terminus. The amino fragment contains a region that determines substrate recognition specificity, and the carboxy terminus contains plant secondary product glycosyltransferases (PSPG) motifs that are important for glycosylation activity. As a result, the recombinant UGT72E2 / 3-1 gene of the present invention comprises an amino fragment comprising amino acids 1 to 344 of the amino terminus of UGT72E2 and a carboxy comprising amino acids 345 to 481 of the carboxy terminus of UGT72E3. The gene encoding the fragment was prepared by linking. The recombinant UGT72E2 / 3-2 gene also encodes an amino fragment comprising amino acids 1 to 240 of the amino terminus of UGT72E2 and a carboxy fragment comprising amino acids 241 to 481 of the carboxy terminus of UGT72E3. It was prepared by connecting.

The gene of the present invention may be DNA or RNA encoding a recombinant sugar transfer enzyme UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. DNA may be a coding strand or a non-coding strand.

The present invention also provides a recombinant vector comprising the gene.

The term “recombinant” 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, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

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

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

Preferred examples of plant expression vectors are Ti-plasmid vectors capable of transferring some of their so-called T-regions to plant cells when present in a suitable host such as Agrobacterium tumefiaciens . Another type of Ti-plasmid vector (see EP 0 116 718B1) is currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. . A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, ampicillin, G418, bleomycin, hygromycin, chloramphenicol There is a gene, but is not limited thereto.

In the 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 a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. "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. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminator of the octopine gene of Bacterium tumerfaciens ( Agrobacterium tumefaciens ), but is not limited thereto.

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

When transforming the recombinant vector of the present invention into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. The host cell is preferably a plant cell.

Methods of transporting the vector of the present invention into the host cell include injection of the vector into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment and the like. can do.

The present invention also transforms plant cells with the recombinant vector to overexpress the gene encoding the UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein. coniferin) provides a method for increasing synthesis.

The method of increasing the synthesis of coniferin of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, for example, Agrobacterium tumefaciens ( A. tumefaciens ) May be mediated by.

In a method according to an embodiment of the present invention, the gene encoding the UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc protein may be SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

The present invention also provides a method for transforming a plant cell with the recombinant vector; and regenerating the plant from the transformed plant cell. It provides a manufacturing method.

The method of the present invention comprises the step of transforming plant cells with the recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumerfaciens. The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

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

In the method according to an embodiment of the present invention, the transformed plant with increased coniferin synthesis preferably increases coniferin synthesis in leaves or roots, and most preferably coniferin synthesis in leaves. This is not restrictive.

The present invention also provides a transgenic plant and its transformed seed having increased coniferin synthesis compared to the wild type produced by the above production method.

In the plant according to an embodiment of the present invention, the plant is preferably Arabidopsis, tobacco, eggplant, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, Chinese cabbage, cabbage, mud, watermelon, melon, cucumber pumpkin, gourd, strawberry, soybean, mung bean, green beans and peas can be dicotyledonous plants, and most preferably may be Arabidopsis, but is not limited thereto.

The present invention also provides a gene encoding the recombinant glycotransferase UGT72E2 / 3-1-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or the recombinant glycotransferase UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 4 It provides a composition for enhancing coniferin synthesis in a plant containing a recombinant vector containing an active ingredient. The composition encodes a recombinant glycotransferase UGT72E2 / 3-1-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or the recombinant glycotransferase enzyme UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 4 as an active ingredient It includes a recombinant vector containing a gene, it is possible to increase the plant's conifer synthesis by transforming the recombinant vector to the plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

1. Preparation of UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc Recombinant Glycotransferase

The present inventors have reported the ability to transfer sugars to cinafil alcohol, a precursor of sirin, or structurally similar coniferyl alcohol, among the 100 sugar transfer enzymes of Arabidopsis, the UGT72E clade. The enzymatic properties of were investigated. The UGT72E family also has structural characteristics similar to those of the common sugar transfer enzymes, with the amino terminal region having a substrate recognition region and the carboxy terminus having an enzymatic active region for transferring sugar from the sugar activated by UDP to the substrate. The carboxy terminal PSPG (Plant Secondary Product Glucosyltransferase) motif has been reported to be important for the activity of glycotransferases derived from plants. The UGT72E family has glycotransferases UGT72E1, UGT72E2, and UGT72E3 with similar sequences.

The inventors of the present invention have excellent substrate specificity for UGT72E2 and sinapyl alcohol, which have excellent sugar transfer efficiency but weak substrate specificity for coniferyl alcohol, which is a precursor of coniferin. Select the UGT72E3 protein, and the UGT72E2 and UGT72E3 proteins are amino fragments containing amino acids 1 to 344 and 1 to 240 amino acids of the amino terminus and 345 to 481 and 241 of the carboxy terminus, respectively. The carboxy fragment contained up to 481 amino acids of the carboxy terminus. The amino fragment contains a region that determines substrate recognition specificity and the carboxy terminus contains a PSPG motif that is important for glycolytic activity. In the boundary region where these two fragments meet, a region for determining substrate specificity and a region for determining metastasis activity are formed. In order to efficiently produce coniferin in plants, both substrate specificity and glycosylation activity are important, so the amino fragment of UGT72E2 is 3/4 or 2/4 of the total, and the UGT72E3 carboxy fragment is a full size 1 containing PSPG motif. UGT72E2 / 3-1 and UGT72E2 / 3-2 were produced by dividing by / 4 or 2/4 and connecting to each other. UGT72E2 / 3-1 was very unstable, making it difficult to produce an overexpressing transformant, and the protein expressed and isolated from Escherichia coli was almost inactive. UGT72E2 / 3-2 is stable in plants and has enhanced substrate specificity for coniferyl alcohol, a precursor of coniferin, but has shown a disadvantage that the enzyme activity is reduced than that of UGT72E2 (FIGS. 1 and 2). Thus, 42 amino acid sequences including three myc effopes were added to the carboxy terminus of the recombinant protein, thereby completing new recombinant glycotransferases UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc.

2. UGT72E2 / 3-1-myc or UGT72E2 / 3-2-myc overexpressing transformants

UGT72E2 gene isolated from Arabidopsis and recombinant UGT72E2 / 3-1, UGT72E2 / 3-2 and myc epitope newly prepared in the present invention UGT72E2-myc, UGT72E2 / 3-1-myc and UGT72E2 / 3-2- A binary vector was constructed to control the coding region of the myc gene by the CaMV35S promoter and the super promoter (Plant Physiol. 2007 145 (4): 1294-1300), and then the vector was converted to Agrobacterium tumefaciens EHA105. The bacteria were then transformed by the in planta method using the bacteria.

Since the phenotype of the transformant is greatly influenced by the expression level of the transgene introduced, the transgene derived from the Arabidopsis herb from the phosphinothricin herbicide-resistant Arabidopsis transformants is stably inserted into the genome. Investigations were carried out using the RT-PCR method. In this case, in order to specifically amplify the introduced gene, a UGT72E gene specific forward primer (5'-GGTTGGAGCTCGACGTTGGAAAGCGTC-3 ', SEQ ID NO: 5) and a reverse primer (5'-TTAAAGCAGGGCATGCCTGC-3', specific for the 3 'UTR region of the vector, SEQ ID NO: 6) combination was used. To correct the relative amount of RNA, the ACTIN1 gene, which is constantly expressed constantly, was used as a reference gene. Of the 15 transformants for each gene examined, the transformant lines with similar expression of UGT72E2, UGT72E2 / 3-1 and UGT72E2 / 3-2 genes were finally identified. In addition, Western blot method using a single Myc antibody was finally confirmed a line of transformants similarly producing UGT72E2-myc, UGT72E2 / 3-1-myc and UGT72E2 / 3-myc protein.

Example 1 Quantitative HPLC Analysis of Coniferin and Syringin Production in Leaves of UGT72E2 and UGT72E2 / 3-2 Overexpressing Transformants

Generally monomonolol produced by activation of the phenylpropaneoid synthesis pathway of the leaves of the genus plants is mostly coniferyl alcohol and is converted to coniferin by sugar transfer enzymes. Very few coniferyl alcohols are converted to sinapyl alcohol, a precursor of sirin, by enzymatic action, and the conversion to sirin is very low due to the activity of low sugar transfer enzymes. Most cinafil alcohols are converted to cinafil esters, which absorb ultraviolet light and serve to protect the leaves. In the plants, the concentration of coniferyl alcohol is higher than that of cinafil alcohol, so in the Arabidopsis overexpressing UGT72E2, coniferin seems to be produced more specifically than syringin. Increase also (Figure 2). These results indicate that UGT72E2 has high reactivity with coniferyl alcohol and cinifil alcohol.

Example 2 Quantitative HPLC Analysis of Coniferin and Syringin Production in Leaves of UGT72E2-myc, UGT72E2 / 3-1-myc, and UGT72E2 / 3-2-myc Overexpressing Transformants

The production of coniferin and syringes in the leaves of UGT72E2-myc (there were no significant differences in enzymatic properties between UGT72E2 and UGT72E2-myc), UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc overexpressing transformants. As a result of analysis, it was confirmed that the production amount of syringes of UGT72E2-myc, UGT72E2 / 3-1-myc, and UGT72E2 / 3-2-myc was insufficient. However, UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc showed about 4 times higher coniferin production rate than UGT72E2-myc (FIG. 4).

In addition, the production of syringes by UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc was very low in the presence of sufficient cinafil alcohol, whereas the production of syringe by UGT72E2-myc was greatly increased (FIG. 5). . These results indicate that UGT72E2 / 3-1-myc and UGT72E2 / 3-2-myc have higher substrate specificity for coniferyl alcohol than UGT72E2-myc.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Gene encoding recombinant UGTase protein increasing coniferin          synthesis in plant and uses <130> PN19006 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1574 <212> DNA <213> Artificial Sequence <220> <223> UGT72E2 / 3-1-myc <400> 1 atgcatatca caaaaccaca cgccgccatg ttttccagtc ccggaatggg ccatgtcatc 60 ccggtgatcg agcttggaaa gcgtctctcc gctaacaacg gcttccacgt caccgtcttc 120 gtcctcgaaa ccgacgcagc ctccgctcaa tccaagttcc taaactcaac cggcgtcgac 180 atcgtcaaac ttccatcgcc ggacatttat ggtttagtgg accccgacga ccatgtagtg 240 accaagatcg gagtcattat gcgtgcagca gttccagccc tccgatccaa gatcgctgcc 300 atgcatcaaa agccaacggc tctgatcgtt gacttgtttg gcacagatgc gttatgtctc 360 gcaaaggaat ttaacatgtt gagttatgtg tttatcccta ccaacgcacg ttttctcgga 420 gtttcgattt attatccaaa tttggacaaa gatatcaagg aagagcacac agtgcaaaga 480 aacccactcg ctataccggg gtgtgaaccg gttaggttcg aagatactct ggatgcatat 540 ctggttcccg acgaaccggt gtaccgggat tttgttcgtc atggtctggc ttacccaaaa 600 gccgatggaa ttttggtaaa tacatgggaa gagatggagc ccaaatcatt gaagtccctt 660 ctaaacccaa agctcttggg ccgggttgct cgtgtaccgg tctatccaat cggtccctta 720 tgcagaccga tacaatcatc cgaaaccgat cacccggttt tggattggtt aaacgaacaa 780 ccgaacgagt cggttctcta tatctccttc gggagtggtg gttgtctatc ggcgaaacag 840 ttaactgaat tggcgtgggg actcgagcag agccagcaac ggttcgtatg ggtggttcga 900 ccaccggtcg acggttcgtg ttgtagcgag tatgtctcgg ctaacggtgg tggaaccgaa 960 gacaacacgc cagagtatct accggaaggg ttcgtgagtc gtactagtga tagaggtttc 1020 atgatcccat catgggcacc gcaagctgaa atcctagccc atcaggccgt tggtgggttt 1080 ttaacacatt gtggttggag ctcgacgttg gaaagcgtcc tttgcggcgt tccaatgata 1140 gcgtggccgc ttttcgccga gcagaatatg aacgcggcgt tgcttagcga tgaactggga 1200 atctctgtta gagtggatga tccaaaggag gcgatttcta ggtcgaagat tgaggcgatg 1260 gtgaggaagg ttatggctga ggacgaaggt gaagagatga gaaggaaagt gaagaagttg 1320 agagacacgg cggagatgtc acttagtatt cacggtggtg gttcggcgca tgagtcgctt 1380 tgcagagtca cgaaggagtg tcaacggttt ttggaatgtg tcggggactt gggacgtggt 1440 gctcgcgcgt ccgagcaaaa gctcatttct gaagaggact tgcgcgcgtc cgagcaaaag 1500 ctcatttctg aagaggactt gcgcgcgtcc gagcaaaagc tcatttctga agaggacttg 1560 cgcgcgttat aagc 1574 <210> 2 <211> 523 <212> PRT <213> Artificial Sequence <220> <223> UGT72E2 / 3-1-myc <400> 2 Met His Ile Thr Lys Pro His Ala Ala Met Phe Ser Ser Pro Gly Met   1 5 10 15 Gly His Val Ile Pro Val Ile Glu Leu Gly Lys Arg Leu Ser Ala Asn              20 25 30 Asn Gly Phe His Val Thr Val Phe Val Leu Glu Thr Asp Ala Ala Ser          35 40 45 Ala Gln Ser Lys Phe Leu Asn Ser Thr Gly Val Asp Ile Val Lys Leu      50 55 60 Pro Ser Pro Asp Ile Tyr Gly Leu Val Asp Pro Asp Asp His Val Val  65 70 75 80 Thr Lys Ile Gly Val Ile Met Arg Ala Ala Val Pro Ala Leu Arg Ser                  85 90 95 Lys Ile Ala Ala Met His Gln Lys Pro Thr Ala Leu Ile Val Asp Leu             100 105 110 Phe Gly Thr Asp Ala Leu Cys Leu Ala Lys Glu Phe Asn Met Leu Ser         115 120 125 Tyr Val Phe Ile Pro Thr Asn Ala Arg Phe Leu Gly Val Ser Ile Tyr     130 135 140 Tyr Pro Asn Leu Asp Lys Asp Ile Lys Glu Glu His Thr Val Gln Arg 145 150 155 160 Asn Pro Leu Ala Ile Pro Gly Cys Glu Pro Val Arg Phe Glu Asp Thr                 165 170 175 Leu Asp Ala Tyr Leu Val Pro Asp Glu Pro Val Tyr Arg Asp Phe Val             180 185 190 Arg His Gly Leu Ala Tyr Pro Lys Ala Asp Gly Ile Leu Val Asn Thr         195 200 205 Trp Glu Glu Met Glu Pro Lys Ser Leu Lys Ser Leu Leu Asn Pro Lys     210 215 220 Leu Leu Gly Arg Val Ala Arg Val Pro Val Tyr Pro Ile Gly Pro Leu 225 230 235 240 Cys Arg Pro Ile Gln Ser Ser Glu Thr Asp His Pro Val Leu Asp Trp                 245 250 255 Leu Asn Glu Gln Pro Asn Glu Ser Val Leu Tyr Ile Ser Phe Gly Ser             260 265 270 Gly Gly Cys Leu Ser Ala Lys Gln Leu Thr Glu Leu Ala Trp Gly Leu         275 280 285 Glu Gln Ser Gln Gln Arg Phe Val Trp Val Val Arg Pro Pro Val Asp     290 295 300 Gly Ser Cys Cys Ser Glu Tyr Val Ser Ala Asn Gly Gly Gly Thr Glu 305 310 315 320 Asp Asn Thr Pro Glu Tyr Leu Pro Glu Gly Phe Val Ser Arg Thr Ser                 325 330 335 Asp Arg Gly Phe Met Ile Pro Ser Trp Ala Pro Gln Ala Glu Ile Leu             340 345 350 Ala His Gln Ala Val Gly Gly Phe Leu Thr His Cys Gly Trp Ser Ser         355 360 365 Thr Leu Glu Ser Val Leu Cys Gly Val Pro Met Ile Ala Trp Pro Leu     370 375 380 Phe Ala Glu Gln Asn Met Asn Ala Ala Leu Leu Ser Asp Glu Leu Gly 385 390 395 400 Ile Ser Val Arg Val Asp Asp Pro Lys Glu Ala Ile Ser Arg Ser Lys                 405 410 415 Ile Glu Ala Met Val Arg Lys Val Met Ala Glu Asp Glu Gly Glu Glu             420 425 430 Met Arg Arg Lys Val Lys Lys Leu Arg Asp Thr Ala Glu Met Ser Leu         435 440 445 Ser Ile His Gly Gly Gly Ser Ala His Glu Ser Leu Cys Arg Val Thr     450 455 460 Lys Glu Cys Gln Arg Phe Leu Glu Cys Val Gly Asp Leu Gly Arg Gly 465 470 475 480 Ala Arg Ala Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala                 485 490 495 Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala Ser Glu Gln             500 505 510 Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala Leu         515 520 <210> 3 <211> 1573 <212> DNA <213> Artificial Sequence <220> <223> UGT72E2 / 3-2-myc <400> 3 atgcatatca caaaaccaca cgccgccatg ttttccagtc ccggaatggg ccatgtcatc 60 ccggtgatcg agcttggaaa gcgtctctcc gctaacaacg gcttccacgt caccgtcttc 120 gtcctcgaaa ccgacgcagc ctccgctcaa tccaagttcc taaactcaac cggcgtcgac 180 atcgtcaaac ttccatcgcc ggacatttat ggtttagtgg accccgacga ccatgtagtg 240 accaagatcg gagtcattat gcgtgcagca gttccagccc tccgatccaa gatcgctgcc 300 atgcatcaaa agccaacggc tctgatcgtt gacttgtttg gcacagatgc gttatgtctc 360 gcaaaggaat ttaacatgtt gagttatgtg tttatcccta ccaacgcacg ttttctcgga 420 gtttcgattt attatccaaa tttggacaaa gatatcaagg aagagcacac agtgcaaaga 480 aacccactcg ctataccggg gtgtgaaccg gttaggttcg aagatactct ggatgcatat 540 ctggttcccg acgaaccggt gtaccgggat tttgttcgtc atggtctggc ttacccaaaa 600 gccgatggaa ttttggtaaa tacatgggaa gagatggagc ccaaatcatt gaagtccctt 660 ctaaacccaa agctcttggg ccgggttgct cgtgtaccgg tctatccaat cggtccctta 720 tgcagaccga tacaatcatc cacgaccgat cacccggttt ttgattggtt aaacaaacaa 780 ccaaacgagt cggttctcta catttccttc gggagtggtg gttctctaac ggctcaacag 840 ttaaccgaat tggcgtgggg gctcgaggag agccagcaac ggtttatatg ggtggttcga 900 ccgcccgttg acggctcgtc ttgcagtgat tatttctcgg ctaaaggcgg tgtaaccaaa 960 gacaacacgc cagagtatct accagaaggg ttcgtgactc gtacttgcga tagaggtttc 1020 atgatcccat catgggcacc gcaagctgaa atcctagccc atcaggccgt tggtgggttt 1080 ttaacacatt gtggttggag ctcgacgttg gaaagcgtcc tttgcggcgt tccaatgata 1140 gcgtggccgc ttttcgccga gcagatatga acgcggcgtt gcttagcgat gaactgggaa 1200 tctctgttag agtggatgat ccaaaggagg cgatttctag gtcgaagatt gaggcgatgg 1260 tgaggaaggt tatggctgag gacgaaggtg aagagatgag aaggaaagtg aagaagttga 1320 gagacacggc ggagatgtca cttagtattc acggtggtgg ttcggcgcat gagtcgcttt 1380 gcagagtcac gaaggagtgt caacggtttt tggaatgtgt cggggacttg ggacgtggtg 1440 ctcgcgcgtc cgagcaaaag ctcatttctg aagaggactt gcgcgcgtcc gagcaaaagc 1500 tcatttctga agaggacttg cgcgcgtccg agcaaaagct catttctgaa gaggacttgc 1560 gcgcgttata agc 1573 <210> 4 <211> 523 <212> PRT <213> Artificial Sequence <220> <223> UGT72E2 / 3-2-myc <400> 4 Met His Ile Thr Lys Pro His Ala Ala Met Phe Ser Ser Pro Gly Met   1 5 10 15 Gly His Val Ile Pro Val Ile Glu Leu Gly Lys Arg Leu Ser Ala Asn              20 25 30 Asn Gly Phe His Val Thr Val Phe Val Leu Glu Thr Asp Ala Ala Ser          35 40 45 Ala Gln Ser Lys Phe Leu Asn Ser Thr Gly Val Asp Ile Val Lys Leu      50 55 60 Pro Ser Pro Asp Ile Tyr Gly Leu Val Asp Pro Asp Asp His Val Val  65 70 75 80 Thr Lys Ile Gly Val Ile Met Arg Ala Ala Val Pro Ala Leu Arg Ser                  85 90 95 Lys Ile Ala Ala Met His Gln Lys Pro Thr Ala Leu Ile Val Asp Leu             100 105 110 Phe Gly Thr Asp Ala Leu Cys Leu Ala Lys Glu Phe Asn Met Leu Ser         115 120 125 Tyr Val Phe Ile Pro Thr Asn Ala Arg Phe Leu Gly Val Ser Ile Tyr     130 135 140 Tyr Pro Asn Leu Asp Lys Asp Ile Lys Glu Glu His Thr Val Gln Arg 145 150 155 160 Asn Pro Leu Ala Ile Pro Gly Cys Glu Pro Val Arg Phe Glu Asp Thr                 165 170 175 Leu Asp Ala Tyr Leu Val Pro Asp Glu Pro Val Tyr Arg Asp Phe Val             180 185 190 Arg His Gly Leu Ala Tyr Pro Lys Ala Asp Gly Ile Leu Val Asn Thr         195 200 205 Trp Glu Glu Met Glu Pro Lys Ser Leu Lys Ser Leu Leu Asn Pro Lys     210 215 220 Leu Leu Gly Arg Val Ala Arg Val Pro Val Tyr Pro Ile Gly Pro Leu 225 230 235 240 Cys Arg Pro Ile Gln Ser Ser Thr Thr Asp His Pro Val Phe Asp Trp                 245 250 255 Leu Asn Lys Gln Pro Asn Glu Ser Val Leu Tyr Ile Ser Phe Gly Ser             260 265 270 Gly Gly Ser Leu Thr Ala Gln Gln Leu Thr Glu Leu Ala Trp Gly Leu         275 280 285 Glu Glu Ser Gln Gln Arg Phe Ile Trp Val Val Arg Pro Pro Val Asp     290 295 300 Gly Ser Ser Cys Ser Asp Tyr Phe Ser Ala Lys Gly Gly Val Thr Lys 305 310 315 320 Asp Asn Thr Pro Glu Tyr Leu Pro Glu Gly Phe Val Thr Arg Thr Cys                 325 330 335 Asp Arg Gly Phe Met Ile Pro Ser Trp Ala Pro Gln Ala Glu Ile Leu             340 345 350 Ala His Gln Ala Val Gly Gly Phe Leu Thr His Cys Gly Trp Ser Ser         355 360 365 Thr Leu Glu Ser Val Leu Cys Gly Val Pro Met Ile Ala Trp Pro Leu     370 375 380 Phe Ala Glu Gln Asn Met Asn Ala Ala Leu Leu Ser Asp Glu Leu Gly 385 390 395 400 Ile Ser Val Arg Val Asp Asp Pro Lys Glu Ala Ile Ser Arg Ser Lys                 405 410 415 Ile Glu Ala Met Val Arg Lys Val Met Ala Glu Asp Glu Gly Glu Glu             420 425 430 Met Arg Arg Lys Val Lys Lys Leu Arg Asp Thr Ala Glu Met Ser Leu         435 440 445 Ser Ile His Gly Gly Gly Ser Ala His Glu Ser Leu Cys Arg Val Thr     450 455 460 Lys Glu Cys Gln Arg Phe Leu Glu Cys Val Gly Asp Leu Gly Arg Gly 465 470 475 480 Ala Arg Ala Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala                 485 490 495 Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala Ser Glu Gln             500 505 510 Lys Leu Ile Ser Glu Glu Asp Leu Arg Ala Leu         515 520 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggttggagct cgacgttgga aagcgtc 27 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttaaagcagg gcatgcctgc 20

Claims (10)

A recombinant glycotransferase protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The gene encoding the recombinant sugar transfer enzyme protein of claim 1. According to claim 2, wherein the gene is a gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. A method of increasing coniferin synthesis in a plant compared to a wild type comprising transforming a plant cell with the recombinant vector of claim 4, thereby overexpressing a gene encoding a recombinant translocation enzyme protein. Transforming plant cells with the recombinant vector of claim 4; and Regenerating plants from the transformed plant cells. The method of producing a transgenic plant with increased coniferin synthesis compared to the wild type. Transgenic plant with increased coniferin synthesis compared to wild type prepared by the method of claim 7. Transformed seed of the plant according to claim 8. A recombinant vector comprising a gene encoding the recombinant sugar transfer enzyme UGT72E2 / 3-1-myc protein consisting of the amino acid sequence of SEQ ID NO: 2 or the recombinant sugar transfer enzyme UGT72E2 / 3-2-myc protein consisting of the amino acid sequence of SEQ ID NO: 4 Composition for enhancing the synthesis of coniferin in plants containing as an active ingredient.

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