CN108359652B

CN108359652B - Glycosyltransferase and application thereof

Info

Publication number: CN108359652B
Application number: CN201710061097.0A
Authority: CN
Inventors: 肖友利; 尤文静
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2021-09-03
Anticipated expiration: 2037-01-25
Also published as: CN108359652A

Abstract

The glycosyltransferase and its application are disclosed. In particular, the present invention provides a polypeptide selected from the group consisting of: (1) 2, a polypeptide shown as SEQ ID NO; and (2) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 and a sequence that promotes the expression, secretion and/or purification of the amino acid sequence shown in SEQ ID NO. 2. The invention also relates to related polynucleotide sequences, nucleic acid constructs, genetically engineered host cells, and uses. The invention further improves the biosynthesis route of stevioside; can be used for industrialization for improving stevioside content by transgenic technology and synthetic biology.

Description

Glycosyltransferase and application thereof

Technical Field

The present invention relates to glycosyltransferases and their uses.

Background

Stevia rebaudiana is native to paraguay, south america, and is a perennial herb of the family compositae. Stevioside, the component extracted from the leaves of stevia rebaudiana Bertoni with the highest content, is a general term for various steviosides. Stevioside accounts for 60% -70% of all sugars in the leaves of stevia rebaudiana, and stevioside (stevioside) and Rebaudioside (Rebaudioside) a are the most important components in the leaves of stevia rebaudiana. Stevia sugar has a sweetness far exceeding that of sucrose, and is the most ideal substitute for sucrose as a low calorie sweetener. Stevia sugar is used in food industry as sweetener, also in medicine industry, has effects of lowering blood pressure and lowering blood sugar, and also has good therapeutic effect on hyperlipemia patients.

The most industrialized production method of stevioside is to extract stevia leaves and refine the stevia leaves, and the extraction of stevioside is divided into two methods, namely a water extraction method and an organic solvent extraction method.

In recent years, with the continuous development of the emerging field of synthetic biology, related genes in a metabolic pathway for synthesizing stevioside are assembled through a transgenic technology, and an engineering strain containing a stevioside biosynthetic pathway is constructed to improve the research and industrialization of stevioside content, so that the method has a good promoting effect on the improvement of the stevioside yield.

Disclosure of Invention

In a first aspect the invention provides a polypeptide selected from the group consisting of:

(1) 2, a polypeptide shown as SEQ ID NO; and

(2) polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 and the amino acid or amino acid sequence promoting the expression, secretion and/or purification of the amino acid sequence shown in SEQ ID NO. 2.

In a second aspect, the invention provides a polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence encoding a polypeptide according to the first aspect of the invention;

(2) a polynucleotide sequence complementary to the polynucleotide sequence of (1); and

(3) a fragment of 10 to 40 bases in length of the polynucleotide sequence of (1) or (2).

In one or more embodiments, the polynucleotide sequence is selected from the group consisting of:

(a) 1, or a polynucleotide sequence shown in SEQ ID NO;

(b) 1, the complement of SEQ ID NO; and

(c) 1 or a fragment of the complementary sequence thereof of 10 to 40 bases in length.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence according to the second aspect of the invention.

In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

In a fourth aspect, the invention provides a genetically engineered host cell which:

(1) expressing a polypeptide according to the first aspect of the invention; and/or

(2) Comprising the nucleic acid construct of the third aspect of the invention.

In one or more embodiments, the host cell further (a) expresses one or more of the polypeptides of

SEQ ID NOs

4, 6, 8, and 10, and/or (b) contains one or more of the expression vectors expressing

SEQ ID NOs

4, 6, 8, and 10.

A fifth aspect of the invention provides a method of synthesizing steviol-19-O-stevioside or rubusoside, the method comprising the step of glycosylating the substrate steviol with one or more of the polypeptides of SEQ ID Nos. 4, 6, 8 and 10.

A sixth aspect of the present invention provides a method for the synthesis of steviol-13-O-stevioside, which comprises the step of glycosylating the substrate steviol with a polypeptide represented by SEQ ID NO. 6 and/or 8.

In a seventh aspect, the present invention provides a method for synthesizing rebaudioside a, the method comprising the step of glycosylating a stevia glycoside substrate with a polypeptide according to the first aspect of the present invention.

An eighth aspect of the present invention provides a method of constructing a transgenic plant, the method comprising:

(1) providing an agrobacterium carrying an expression vector expressing the polypeptide of the invention;

(2) contacting a plant cell or tissue or organ with the agrobacterium of step (1) such that the coding sequence is transferred into the plant cell and integrated into the chromosome of the plant cell;

(3) selecting a plant cell or tissue transformed with the coding sequence; and

(4) regenerating the plant cell or tissue of step (3) into a plant.

In a ninth aspect, the present invention provides a compound X having the structure shown below:

in one or more embodiments, the compound X is used as a sweetener.

A tenth aspect of the present invention provides a process for preparing compound X as described above, which comprises the step of glycosylating rebaudioside I with SEQ ID NO 10 or a mutant thereof which retains glycosyltransferase activity.

The invention also provides one or more of SEQ ID NO 2 or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 4 or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 6 or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 8 or a mutant thereof retaining glycosyltransferase activity, and SEQ ID NO 10 or a mutant thereof retaining glycosyltransferase activity, a coding sequence, an expression vector thereof, or the genetically engineered host cell of the invention for use in preparing stevioside, or for use in preparing rebaudioside A, or for use in preparing compound X.

In one or more embodiments, the host cell expresses one or more of SEQ ID NO 2 or a mutant thereof which retains glycosyltransferase activity, SEQ ID NO 4 or a mutant thereof which retains glycosyltransferase activity, SEQ ID NO 6 or a mutant thereof which retains glycosyltransferase activity, SEQ ID NO 8 or a mutant thereof which retains glycosyltransferase activity, and SEQ ID NO 10 or a mutant thereof which retains glycosyltransferase activity.

In one or more embodiments, the genetically engineered host cell expresses at least SEQ ID NO. 2 or a mutant thereof that retains glycosyltransferase activity, while also expressing SEQ ID NO. 4, 6 and 8 or mutants thereof that retain glycosyltransferase activity. In certain embodiments, the host cell further expresses SEQ ID NO. 10 or a mutant thereof that retains glycosyltransferase activity. In one or more embodiments, the host cell further expresses other glycosyltransferases in the stevia glycosides synthesis pathway.

The invention also provides one or more of SEQ ID NO 2 or a mutant thereof retaining the glycosyltransferase activity, SEQ ID NO 4 or a mutant thereof retaining the glycosyltransferase activity, SEQ ID NO 6 or a mutant thereof retaining the glycosyltransferase activity, SEQ ID NO 8 or a mutant thereof retaining the glycosyltransferase activity, and SEQ ID NO 10 or a mutant thereof retaining the glycosyltransferase activity, a coding sequence, an expression vector thereof, or the use of the genetically engineered host cell of the invention in the preparation of a compound of the formula:

in the formula (I), the compound is shown in the specification,

R₁selected from: H. glc β 1-, Glc β 1-3Glc β 01-, Glc β 11-2Glc β 21-, Glc β 31-2(Glc β 1-3) -Glc β 1-, Glc β 1-2(Glc β 1-6) -Glc β 1-, and Glc β 1-3(Glc β 1-6) -Glc β 1-; and

R₂selected from: H. glc β 1-, Glc β 1-2Glc β 1-, and Glc β 1-2(Glc β 1-3) -Glc β 1-.

In one or more compounds, the compound is selected from: steviol, steviol-13-O-stevioside, steviol-19-O-stevioside, rubusoside, steviolbioside, stevioside, rebaudioside A, rebaudioside I, rebaudioside D, rebaudioside M2 and compound X.

Drawings

FIG. 1: and (3) amplifying the electrophoresis result of the glycosyltransferase related gene segment by taking the cDNA as a template.

FIG. 2: and (4) carrying out electrophoresis detection on the SrUGT85A8 by SDS-PAGE. M: protein Marker, CL: cell lysate, CP: disrupted cell pellet, CS: disrupted cell supernatant, FT: supernatant, WB: unbound protein eluted without imidazole buffer, 1-6: 15mM, 25mM, 50mM, 100mM, 250mM, 250mM imidazole.

FIG. 3: SDS-PAGE electrophoresis detection of SrUGT76G 2. M: protein Marker, CL: cell lysate, CS: disrupted cell supernatant, CP: disrupted cell pellet, FT: supernatant, WB: unbound protein eluted without imidazole buffer, 1-4: 15mM, 50mM, 100mM, 250mM imidazole.

FIG. 4: electrophoretic detection of SDS-PAGE of AtUGT73C 1. M: protein Marker, CS: disrupted cell supernatant, CP: disrupted cell pellet, FT: supernatant, 1-4: 25mM, 50mM, 100mM, 250mM imidazole.

FIG. 5: electrophoretic detection of SDS-PAGE of AtUGT73C 5. M: protein Marker, CL: cell lysate, CS: disrupted cell supernatant, CP: disrupted cell pellet, FT: supernatant, WB: unbound protein eluted without imidazole buffer, 1-3: 15mM, 50mM, 250mM imidazole.

FIG. 6: SDS-PAGE electrophoresis detection of SrUGT76G 1. M: protein Marker, CL: cell lysate, CP: disrupted cell pellet, CS: disrupted cell supernatant, FT: supernatant, WB: unbound protein eluted without imidazole buffer, 1-6: 15mM, 25mM, 50mM, 100mM, 250mM, 250mM imidazole.

FIG. 7: HPLC analysis of the glycosyltransferases SrUGT85A8, AtUGT73C1 and AtUGT73C1 reacted with Steviol. In the figure, peak a is at 22.5min, indicating steviol; peak B, at 12.5min, indicates steviol-13-O-stevioside; the C peak is at 11.3min, indicating formation of steviol-19-O-stevioside; peak D was at 5.3min, indicating rubusoside.

FIG. 8: HPLC analysis of glycosyltransferases SrUGT85A8, AtUGT73C1 and AtUGT73C1 reacted with steviol-19-O-stevioside. In the figure, peak C is at 11.3min, indicating the production of steviol-19-O-stevioside; peak D was at 5.3min, indicating rubusoside.

FIG. 9: HPLC analysis of the reaction of the glycosyltransferase SrUGT76G2 with stevioside. In the figure, the peak E is at 10.7min, indicating stevioside; peak F was at 10.2min, indicating rebaudioside a.

FIG. 10: LC/MS analysis the mass spectrum result of the reaction of AtUGT73C1 with steviol. A) Full wavelength scan of the AtUGT73C1 reaction with steviol; b) mass spectrum results of reaction products of AtUGT73C1 and steviol, namely steviol-13-O-stevioside and steviol-19-O-stevioside, are shown in the specification, wherein the mass spectrum results are shown in the specification of [ M + Na [)]⁺Theoretical value 503.2615, actual value 503.2507; c) mass spectrum result of AtUGT73C1 and steviol reaction product rebaudioside A, [ M + Na ]]⁺Theoretical 665.3144 and actual 665.3003.

FIG. 11: LC/MS analysis of Mass Spectrometry results of the reaction of AtUGT73C5 with steviol a) full wavelength Scan of the reaction of AtUGT73C5 with steviol; b) AtUGT73C5 and Steviol reaction products including Steviol-13-O-stevioside, Steviol-19-O-steviosideThe result of mass spectrometry of [ M + Na ]]⁺Theoretical value 503.2615, actual value 503.2507; c) mass spectrum result of AtUGT73C5 and steviol reaction product rebaudioside A, [ M + Na ]]⁺Theoretical 665.3144 and actual 665.3003.

FIG. 12: and analyzing mass spectrum results of the reaction of SrUGT85A8 and steviol by LC/MS. a) A full wavelength scan of SrUGT85A8 reacted with steviol; b) mass spectrum result of reaction product of SrUGT85A8 and steviol, namely steviol-19-O-stevioside, [ M + Na ]]⁺Theoretical 503.2615 and actual 503.2507.

FIG. 13: LC/MS analysis of mass spectrum results of SrUGT76G2 reaction with steviol glycosides. a) Full wavelength scanning, chromatogram and product molecular weight extracted ion flow of SrUGT76G2 and stevioside reaction; b) mass spectrometry results of the reaction product of SrUGT76G2 with steviol glycoside rebaudioside a, [ M + NH₄]⁺Theoretical 984.4646 and actual 984.4649.

FIG. 14: and analyzing mass spectrum results of the reaction of SrUGT76G1 and rebaudioside A by LC/MS. a) Full-wavelength scanning and chromatogram of SrUGT76G1 and rebaudioside A reaction and ion flow diagrams of molecular weight extraction of a penta-stevioside product and three hexa-stevioside products; b) mass spectrometry results of a stevia pentasaccharide product from the reaction of SrUGT76G1 with rebaudioside a, [ M + NH4]⁺Theoretical 1146.5174 and actual 1146.5150. Mass spectrometry results of three stevia products from the reaction of SrUGT76G1 with rebaudioside a, [ M + NH₄]⁺Theoretical 1380.5703, actual 1308.5662, 1308.5661 and 1308.5648.

FIG. 15: LC/MS analysis of mass spectra results of SrUGT76G1 reacted with rebaudioside I. a) Full wavelength scan, chromatogram of SrUGT76G1 reaction with rebaudioside I and ion flow graph of molecular weight extraction of substrate rebaudioside I and product X; b) mass spectrometry results of SrUGT76G1 and rebaudioside I reaction product X, [ M + NH ]₄]⁺Theoretical 1380.5703 and actual 1308.5617.

FIG. 16: steviol and steviol glycoside structures.

FIG. 17: biosynthetic metabolic pathway of stevioside.

Detailed Description

The invention respectively constructs expression vectors of glycosyltransferases SrUGT85A8, SrUGT76G1, SrUGT76G2, AtUGT73C1 and AtUGT73C5, and carries out in-vitro enzyme activity test. It is found that the glycosyltransferases AtUGT73C1 and AtUGT73C5 from Arabidopsis thaliana can catalyze Steviol (Steviol) to generate Steviol-19-O-stevioside (Steviol-19-O-Glucoside) and Steviol-13-O-stevioside (Steviolmonoside), further catalyze Steviol-19-O-stevioside to generate Rubusoside (Rubusoside), and the catalytic efficiency is relatively high for the plant source UGT; stevia derived SrUGT85A8 glycosylates the carboxyl groups of steviol to produce steviol-19-O-stevioside, while the glycosyltransferase SrUGT76G2 can catalyze stevioside to produce rebaudioside A, the glycosyltransferase SrUGT76G1 can catalyze rebaudioside A to produce rebaudioside D, further to produce rebaudioside M2, and SrUGT76G1 can also catalyze rebaudioside I to further glycosylate to produce new compound X. Thus, the present invention has been completed. These findings of the present invention further improve the biosynthetic pathway of steviol glycosides; can be used for industrialization for improving stevioside content by transgenic technology and synthetic biology.

Specifically, the present invention provides a glycosyltransferase (UGT) having an amino acid sequence as set forth in

SEQ ID NO

2, 4, 6, 8 or 10. The invention also includes polypeptides obtained by conservative substitution of one or several amino acids with similar or analogous properties on the basis of the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8 or 10. Such conservative substitutions do not generally alter the function of the protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

The polypeptide of the present invention thus includes mutants of the amino acid sequence shown in

SEQ ID NO

2, 4, 6, 8 or 10, i.e. polypeptides derived from

SEQ ID NO

2, 4, 6, 8 or 10 by substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in

SEQ ID NO

2, 4, 6, 8 or 10 while retaining the lysine decarboxylase activity possessed by the amino acid sequence shown in

SEQ ID NO

2, 4, 6, 8 or 10. The number of the units is usually 10 or less, preferably 8 or less, and more preferably 5 or less.

In certain embodiments, the polypeptides of the invention also include UGT74G1 and mutants thereof that retain functional activity.

Those skilled in the art can determine which amino acid residues in the amino acid sequences shown in

SEQ ID NO

2, 4, 6, 8, 10 and UGT74G1 can be substituted or deleted by using conventional technical means. For example, by aligning sequences from different species, having the same or similar or significantly different activities, it can be determined which amino acid residues in the sequences can be substituted or deleted. Such sequences can be verified for enzymatic activity according to the present invention using methods conventional in the art, including those disclosed herein.

Furthermore, it is well known to those skilled in the art that in gene cloning procedures, it is often necessary to design appropriate cleavage sites, which necessitate the introduction of one or more irrelevant residues at the end of the expressed protein, which do not affect the activity of the protein of interest. As another example, to construct a fusion protein, to facilitate expression of a recombinant protein, to obtain a recombinant protein that is automatically secreted outside of a host cell, or to facilitate purification of a recombinant protein, it is often necessary to add amino acids or amino acid sequences to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, or the proteolytic enzyme site of factor Xa or thrombin or enterokinase, and protein tags that can purify the protein, such as FLAG, HA, HA1, C-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1, and the like. It is understood that the presence of these amino acid sequences does not affect the activity of the resulting polypeptide. Thus, the invention also includes polypeptides having one or more amino acids added to the C-terminus and/or N-terminus of the polypeptides of the invention (including SEQ ID NOs: 2, 4, 6, 8, and 10) that still have glycosyltransferase activity as described herein.

Thus, the invention also includes amino acid sequences having at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to the amino acid sequence set forth in

SEQ ID NO

2, 4, 6, 8, 10 or UGT74G 1. In a more preferred embodiment, such amino acid sequences are also from stevia or Arabidopsis thaliana, preferably having the same or similar glycosyltransferase enzyme activity as SEQ ID NO:2, 4, 6, 8, 10 or UGT74G1, e.g., having the same or similar kinetic parameters (e.g., Km, Kcat and Kcat/Km values) in the same reaction system and under the same reaction conditions.

Sequence identity can be calculated for two sequences aligned by conventional means, for example, using BLASTP provided by NCBI and using default parameters for alignment.

The polypeptides of the invention can be naturally purified products, or chemically synthesized products, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect and mammalian cells).

The present application includes polynucleotide sequences selected from the group consisting of polynucleotide sequences encoding polypeptides of the present invention and complementary sequences. 1, 3, 5, 7 and 9 show exemplary coding sequences for the polypeptides of the invention. The polynucleotide sequences of the invention also include polynucleotide sequences that are highly homologous to the coding and complementary sequences, or polynucleotide sequences that hybridize under stringent conditions, or family gene molecules that are highly homologous to

SEQ ID NOs

1, 3, 5, 7, or 9. The invention also encompasses fragments of 10 to 40 bases, preferably 15 to 30 bases, of a polynucleotide sequence of the invention, which fragments are useful as probes or primers. As used herein, "fragment" refers to a contiguous portion of the full-length sequence.

Sequences encoding the polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The polynucleotide sequence of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis.

The invention also relates to nucleic acid constructs comprising a polynucleotide sequence of the invention and one or more control sequences operably linked to the polynucleotide sequence and directing the expression of the polynucleotide sequence in a host cell under suitable conditions. The polynucleotide sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to ensure expression of the polypeptide. Manipulation of the polynucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence comprises transcriptional regulatory sequences linked to the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of promoter sequences suitable for use in the present invention include the 35S promoter and the cspA promoter, among others.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The check-in sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino acid terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5' end of the nucleotide sequence coding sequence may inherently contain a native signal peptide coding region. Alternatively, the 5' end of the coding sequence may comprise a signal peptide coding region foreign to the coding region. Optionally, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide.

The invention also relates to cloning or expression vectors comprising a polynucleotide of the invention. These vectors may contain various regulatory sequences as described previously.

The expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence of interest. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may comprise any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids, or a transposon, which together contain the total DNA to be introduced into the genome of the host cell, may be used.

The vectors of the invention preferably comprise one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or the like cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The vectors of the present invention preferably contain elements that permit integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase the yield of the gene product. An increase in the copy number of a polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and, thus, additional copies of the polynucleotide can be screened for by culturing the cells in the presence of the appropriate selectable agent.

The vectors of the present invention preferably comprise a synthetic sequence containing multiple restriction enzyme recognition sites to provide multiple sites or insertion schemes for foreign DNA. The expression vector of the invention preferably contains small peptides with 6 consecutive histidine sequences, which is beneficial to the extraction and purification of protein.

Cloning vectors containing the polynucleotide sequences of the present invention are useful for replicating a sufficient number of plasmids of interest. Therefore, the cloning vector of the present invention has strong self-replicating elements such as replication initiation sites and the like. Typically, the cloning vectors of the present invention do not have expression elements.

The present invention also relates to recombinant host cells containing a polynucleotide of the present invention which are used for the recombinant production of the polypeptide. The vector comprising the polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as an extrachromosomal self-replicating vector as described earlier. The choice of host cell will depend to a large extent on the gene encoding the polypeptide and its source.

The host cell may be a plant cell or a unicellular microorganism or a non-unicellular microorganism. The host cell may be a prokaryotic cell including bacteria of the genera Saccharomyces, Pseudomonas, Bacillus, Enterobacter, Staphylococcus, Streptomyces and Escherichia. In certain embodiments, the host cell is a plant cell, such as a stevia-derived cell or an arabidopsis-derived cell. In certain embodiments, the host cell is escherichia coli or yeast.

In certain aspects, the host cell of the invention is an agrobacterium containing an expression vector that expresses a polypeptide of the invention. The Agrobacterium can be an Agrobacterium known in the art, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes.

Nucleic acid constructs comprising a polynucleotide sequence of the invention can be transferred into host cells using conventional transfection procedures. Transfection is generally divided into transient transfection and stable transfection. The former exogenous DNA/RNA is not integrated into the host chromosome, so multiple copy numbers can be present in a host cell, resulting in high levels of expression, but usually only lasting a few days. In stable transfection, the foreign DNA may be either integrated into the host chromosome or may be present as an episome. The technical means of transfection include chemical transfection such as DEAE-dextran method, calcium phosphate method and artificial liposome method, and physical transfection such as microinjection, electroporation, gene gun, etc.

It is to be understood that the host cell of the invention may express only SEQ ID NO 2 and/or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 4 and/or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 6 and/or a mutant thereof retaining glycosyltransferase activity, or SEQ ID NO 8 and/or a mutant thereof retaining glycosyltransferase activity, or SEQ ID NO 10 and/or a mutant thereof retaining glycosyltransferase activity, or may express SEQ ID NO 2 and/or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 4 and/or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 6 and/or a mutant thereof retaining glycosyltransferase activity, SEQ ID NO 8 and/or a mutant thereof retaining glycosyltransferase activity and SEQ ID NO 10 and/or a mutant thereof Any two, any three or all four of the mutants retaining glycosyltransferase activity, for example, simultaneously expressing SEQ ID NO 6 and/or a mutant thereof retaining glycosyltransferase activity with SEQ ID NO 8 and/or a mutant thereof retaining glycosyltransferase activity; or simultaneously expressing SEQ ID NO. 4 and/or a mutant thereof retaining the glycosyltransferase activity, SEQ ID NO. 6 and/or a mutant thereof retaining the glycosyltransferase activity and SEQ ID NO. 8 and/or a mutant thereof retaining the glycosyltransferase activity, or further expressing SEQ ID NO. 2 and/or a mutant thereof retaining the glycosyltransferase activity. In certain embodiments, expression vectors for other enzymes of the stevioside biosynthetic metabolic pathway known in the art, including but not limited to UGT74G1 (accession number: AY345982), may also be transferred into the host cells of the invention.

After obtaining the coding sequence for the polypeptide, the polypeptide of the present invention can be produced by a method comprising: (a) culturing the host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

In the production methods of the present invention, the cells can be cultured in a medium suitable for production of the polypeptide using methods known in the art. For example, a cell may be cultured by shake flask culture and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. Cultivation takes place in a suitable medium comprising carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or may be prepared according to the disclosed compositions. If the polypeptide is secreted into the culture medium, the polypeptide can be recovered directly from the culture medium. If the polypeptide is not secreted into the culture medium, it can be recovered from the cell lysate.

The polypeptides described herein can be recovered using methods known in the art. For example, the polypeptide can be recovered from the culture medium by conventional methods, including but not limited to centrifugation, filtration, ultrafiltration, extraction, chromatography, spray drying, freeze drying, evaporation, or precipitation, and the like.

The polypeptides of the invention can be purified by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, size exclusion), electrophoresis (e.g., isoelectric focusing), differential solubility (e.g., salting-out precipitation), SDS-PAGE, or extraction to obtain a substantially pure polypeptide.

The polypeptides of the invention may be used in the preparation of stevioside, for example, in the biosynthesis of steviol-13-O-stevioside, steviol-19-O-stevioside, rubusoside, stevioside, and/or rebaudioside A. For example, engineered strains containing the stevioside biosynthetic pathway can be constructed using the expression vectors of the invention, and one or more products of the stevioside biosynthetic pathway can be produced via the engineered strains. FIG. 17 shows the biosynthetic metabolic pathway of stevioside. Thus, for example, one or more of

SEQ ID NOs

4, 6, 8 or mutants thereof which retain glycosyltransferase activity may be used in the synthesis of steviol-19-O-stevioside or rubusoside; one or more of

SEQ ID NOs

6, 8 or mutants thereof retaining glycosyltransferase activity may be used in the synthesis of steviol-13-O-stevioside. Whereas SEQ ID NO 10 and/or mutants thereof retaining glycosyltransferase activity can be used to glycosylate rebaudioside A to synthesize rebaudioside D, rebaudioside M2, rebaudioside M and rebaudioside I. 2 and/or mutants thereof which retain glycosyltransferase activity are useful for glycosylating stevia glycosides to synthesize rebaudioside A and for glycosylating rebaudioside I to synthesize compound X.

Thus, the engineered strain of the invention may express one or more of

SEQ ID NO

2, 4, 6, 8, 10 or mutants thereof retaining glycosyltransferase activity, or one or more expression vectors comprising the same which may express

SEQ ID NO

2, 4, 6, 8, 10 or mutants thereof retaining glycosyltransferase activity. In certain embodiments, the engineered strain of the invention expresses at least SEQ ID NO 2 or a mutant thereof which retains glycosyltransferase activity, and also expresses one or more of

SEQ ID NO

4, 6, 8, 10 or a mutant thereof which retains glycosyltransferase activity, such as

SEQ ID NO

4, 6 and 8, or a mutant thereof which retains glycosyltransferase activity, or further expresses SEQ ID NO 10 or a mutant thereof which retains glycosyltransferase activity. Or the engineering strain at least contains an expression vector capable of expressing SEQ ID NO. 2 or a mutant thereof retaining the glycosyltransferase activity, and also contains one or more of expression vectors for expressing SEQ ID NO. 4, 6, 8 and 10 or mutants thereof retaining the glycosyltransferase activity, such as expression vectors for expressing SEQ ID NO. 4, 6 and 8 or mutants thereof retaining the glycosyltransferase activity, or further contains an expression vector for expressing SEQ ID NO. 10 or mutants thereof retaining the glycosyltransferase activity.

Preferably, other enzymes in the stevioside biosynthetic pathway, such as UGT74G1, are also expressed in the engineered strains of the invention. Examples of engineered strains that may be used in the present invention include, but are not limited to, E.coli, yeast, and the like.

In certain embodiments, the present invention relates to a method of constructing a transgenic plant, the method comprising:

(2) contacting a plant cell or tissue or organ with the Agrobacterium of step (1) such that the coding sequence for the polypeptide of the invention is transferred into the plant cell and integrated into the plant cell chromosome;

(3) selecting a plant cell or tissue transformed with the coding sequence; and

(4) regenerating the plant cell or tissue of step (3) into a plant.

It is understood that the expression vector transferred into the plant cell may express one polypeptide of the present invention, or may express two or more polypeptides. Thus, one or more expression vectors can be transferred into the plant cells to enable the constructed transgenic plants to synthesize one or more, or all, of the products of the stevioside biosynthetic pathway.

Preferably, such transgenic plants are naturally synthetic stevioside plants, such as stevia rebaudiana. The transgenic method of the invention can improve the yield of stevioside in the plant. Alternatively, such transgenic plants may be plants suitable for the industrial production of stevioside, and after transfer of a vector expressing a polypeptide of the invention by a transgenic method of the invention, may be capable of expressing a polypeptide of the invention and biosynthesizing one or more or all of the products of the stevioside biosynthetic pathway.

In certain embodiments, the invention provides plant tissue comprising cells comprising an expression vector of the invention expressing one or more polypeptides of the invention. The plant tissue may be a variety of plant tissues known in the art, such as callus, seed, or a portion of a plant. In certain embodiments, a portion of the plant is not regenerated into a plant. For example, the plant tissue of the invention is a leaf of stevia rebaudiana that is enriched in one or more or all of the products of the stevioside biosynthetic pathway. One or more or all of the stevioside biosynthetic pathway in the leaf can be extracted using aqueous and organic solvent extraction methods well known in the art.

In certain embodiments, the present invention also provides compound X having the formula:

the compound X is acid and alkali resistant, soluble in water, and can be used as sweetener. The compound X can be obtained by glycosylating rebaudioside I with SEQ ID NO 10 or a mutant thereof retaining glycosyltransferase activity according to the present invention.

The present invention will be illustrated below by way of specific examples. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (Cold spring harbor laboratory Press, N.Y., USA, 1989) or according to the manufacturer's recommendations. For the use and amounts of the reagents, the conventional use and amounts are used unless otherwise indicated.

Example 1: extraction and detection of total RNA

The clean bench and the ultraviolet lamp are turned on, and the mortar, the key and the scissors are burnt by ethanol. Shearing off 100mg of leaves, grinding the tissues into powder in liquid nitrogen, subpackaging into Ep tubes, adding 1ml of Trizol-x-100 after the liquid nitrogen is volatilized, immediately shaking forcefully or sucking by a pipette for 5-8 times (till no block exists), and standing for 5min at room temperature; extracting with chloroform of equal volume for 2 times, centrifuging at 7500g for 15 min; adding equal volume of pre-cooled isopropanol into the supernatant, mixing, standing at room temperature for 30min, centrifuging at 4 deg.C for 10min at 10000 g; adding 1ml 75% ethanol into the precipitate, cleaning, centrifuging at 4 deg.C 10000g for 10 min; the pellet was dried at room temperature for 10min and dissolved in 25. mu.l of DEPC treated water, RNA integrity was checked by 1.0% agarose gel electrophoresis, and the A260, A280 ratios and concentrations were determined using an Eppendorf nucleic acid quantifier. Placing in a refrigerator at-80 deg.C for use.

Example 2: construction of recombinant strains

(1) Reverse transcription to synthesize cDNA

The first complementary strand of stevia mRNA was synthesized using the total RNA prepared in example 1 using the PrimeScript reverse transcription kit supplied by Takara corporation.

(2) Cloning of glycosyltransferase genes

A CDS sequence of stevia glycosyltransferase SrUGT85A8 (the nucleotide sequence is shown as SEQ ID NO:3, and the amino acid sequence is shown as SEQ ID NO: 4), Arabidopsis AtUGT73C1 (the nucleotide sequence is shown as SEQ ID NO:5, and the amino acid sequence is shown as SEQ ID NO: 6), Arabidopsis AtUGT73C5 (the nucleotide sequence is shown as SEQ ID NO:7, and the amino acid sequence is shown as SEQ ID NO: 8), and stevia glycosyltransferase SrUGT76G1 (the nucleotide sequence is shown as SEQ ID NO:9, and the amino acid sequence is shown as SEQ ID NO: 10) is searched from NCBI, and simultaneously, analysis is carried out on stevia transcriptome data to find a new glycosyltransferase with potential functions, which is named as SrUGT76G2 (the nucleotide sequence is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO: 2).

Through alignment analysis, 4 pairs of amplification primers as shown in table 1 were designed using vector NTI software. Is prepared from stevia rebaudiana,

Arabidopsis cDNA was used as a template, and ddH was added thereto in the following system [ 4.0. mu.l of 5 XPisuon buffer, 1.6. mu.l of dNTP (2.5mM), 2. mu.l of each primer (2. mu.M), 0.6. mu.l of DMSO, 0.5. mu.l of cDNA, and 0.2. mu.l of Phusion DNA polymerase₂O to a final volume of 20.0. mu.L) glycosyltransferase gene was amplified, reaction program: pre-denaturation at 98 deg.C for 30s, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min, 30 cycles, final extension at 72 deg.C for 5min, and storage at 16 deg.C. The amplification results were checked by electrophoresis on a 1% agarose gel, and the results are shown in FIG. 1.

The PCR product was purified using the Agarose Gel Fragment Recovery Kit Ver.2.0 from Axygen,

then cloning to pMD19-T vector, screening positive clone and extracting plasmid, sending to Sony corporation for sequencing, obtaining glycosyltransferase related gene sequence.

Table 1: primers for glycosyltransferase amplification (SEQ ID NO: 11-20)

(3) Construction of a glycosyltransferase proenzyme Nuclear expression vector

Taking a glycosyltransferase gene in a T vector as a template, performing PCR amplification by using the primers shown in the table 1, performing electrophoresis detection and purification on an amplification product, performing enzyme digestion for 2 hours at 37 ℃ by using restriction enzyme, and purifying the enzyme digestion product by using a gel recovery kit (Axygen company). SrUGT85A8 was digested with restriction enzymes BamHI and NcoI at 37 ℃ for 2h and ligated to pET-28a (+), SrUGT76G2 was digested with restriction enzymes BamHI and NotI at 37 ℃ for 2h and ligated to pET-28a (+), AtUGT73C1 was digested with restriction enzymes BamHI and NotI at 37 ℃ for 2h and ligated to pET-28a (+), AtUGT73C5 was digested with restriction enzymes BamHI and NotI at 37 ℃ for 2h and ligated to pET-28a (+), and SrUGT76G1 was digested with restriction enzymes BamHI and NotI at 37 ℃ for 2h and ligated to pET-28a (+).

(4) Construction of recombinant strains

Respectively reacting the PCR fragments and the carrier fragments at 16 ℃ for 12h under the action of DNA ligase, and utilizing CaCl as a ligation product₂Coli BL21(DE3) was transformed, and screened on kanamycin-resistant plates, and the clones were subjected to whole-cell PCR detection, and the positive clones were inoculated into LB liquid medium for culture, recombinant plasmids were extracted, and BamHI and NotI double-restriction enzyme validation was performed, and agarose gel electrophoresis was performed to identify the presence of two fragments after complete restriction enzyme, indicating successful construction of expression vectors, and designated pET-28a (+)/SrUGT85A8, pET-28a (+)/SrUGT76G2, pET-28a (+)/AtUGT73C1, pET-28a (+)/AtUGT73C5, and pET-28a (+)/SrUG76G1, respectively.

Example 3: expression of glycosyltransferase genes

The positive clones obtained by screening in example 2 were inoculated into LB liquid medium containing kana resistance (final concentration 100. mu.g/mL), cultured at 37 ℃ for 3-4h at 150r/min, then the density of the cells was measured by a spectrophotometer, IPTG (final concentration 0.2mM) was added at OD600 of about 0.6 to induce expression, 1mL of the cell suspension was put into a 1.5mL centrifuge tube every 2h, the cells were collected by centrifugation, 2 Xloading buffer solutions of equal volume were added, centrifuged for a short time in a boiling water bath for 5min, and analyzed by 10% SDS-PAGE electrophoresis. Compared with the empty vector recombinant strain [ pET-28a (+) ], the recombinant glycosyltransferase expression vector has one more protein band, and the expression amount of the glycosyltransferase gene is gradually increased along with the increase of the induction time.

Example 4: purification and Activity identification of glycosyltransferase Gene expression vectors

(1) Purification of glycosyltransferase Gene expression vectors

Culturing a large amount of recombinant strains, carrying out IPTG induced expression, collecting thalli, using Tris buffer solution (100mM, pH8.0) to resuspend the thalli, dissolving lysozyme, crushing cells by an ice bath squeezer, centrifuging at 18000rpm for 30min, collecting supernatant, adopting Ni-NTA to purify recombinant glycosyltransferase according to His-Tag on fusion protein, respectively eluting target protein by using imidazole solutions with different concentrations, collecting purified liquid, and detecting the purification effect by 10% SDS-PAGE electrophoresis. The results are shown in FIGS. 2 to 5.

(2) Activity identification of glycosyltransferase Gene expression vectors

An enzyme reaction system was prepared in Tris buffer (100mM, pH8.0) so that the final concentrations of the reagents were: BSA (0.01mg/ml), UDPG (2mM), MgCl₂(5mM), substrate (1mM) and each glycosyltransferase purified in step (1) (30. mu.M). The reaction system is placed in a water bath kettle at 37 ℃ for 2 h.

And (4) HPLC detection: the reaction solution was extracted 2 times with 300. mu.l of saturated n-butanol, vortexed, centrifuged at 12000rpm for 10s, the n-butanol phase was concentrated by centrifugation, and then dissolved in 50% MeOH. Phenomenex C18, 4.6X 250mm, 4 μm (p/n 00G-4375-E0); column temperature: 30 ℃; mobile phase A: 0.1% formic acid, organic phase B: acetonitrile; flow rate: 1.0 mL/min; sample introduction amount: 20 mu L of the solution; ultraviolet detection (210 nm).

The results are shown in FIGS. 7-15.

The results of fig. 7 show that stevia rebaudiana SrUGT85A8 glycosylates the carboxyl group of Steviol (Steviol) to generate Steviol-19-O-stevioside (Steviol-19-O-Glucoside), and the structure of Steviol-19-O-stevioside is verified by two-dimensional nuclear magnetic resonance; arabidopsis thaliana AtUGT73C1 and AtUGT73C5 can be glycosylated with Steviol (Steviol) to generate Steviol-19-O-stevioside, Steviol-13-O-stevioside (Steviolmonoside) and Rubusoside (Rubusoside), and the structures of the three products are verified by nuclear magnetism.

The results of FIG. 8 show that stevia SrUGT85A8 is unable to glycosylate steviol-19-O-stevioside; arabidopsis AtUGT73C1 and AtUGT73C5 can glycosylate steviol-19-O-stevioside to generate rubusoside, and the product structure is verified by nuclear magnetism.

The HPLC result of FIG. 9 shows that stevia rebaudiana SrUGT76G2 can be used for glycosylating stevioside to generate rebaudioside A, and the structure of the product is verified by nuclear magnetism.

FIGS. 10-12 show the mass spectra of the LC/MS analysis of the reaction of AtUGT73C1, AtUGT73C5 and SrUGT85A8 with steviol, respectively.

Fig. 13 shows the mass spectrometry results of LC/MS analysis of SrUGT76G2 reacted with steviol glycosides.

FIG. 14 shows the mass spectrometry results of LC/MS analysis of the reaction of SrUGT76G1 with rebaudioside A.

FIG. 15 shows the mass spectrometry results of LC/MS analysis of the reaction of SrUGT76G1 with rebaudioside I.

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> glycosyltransferase and use thereof

<130> 168067

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 1432

<212> DNA

<213> Stevia rebaudiana (Bertoni) Hemsl)

<400> 1

ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg agtgcggccg 60

cttagaatga cgaaatataa tgaatgagag actccaatga ttcattagaa gatccactta 120

tcatcaaaga atcatcaacc ttttgtttca aaacttttac cctctctcta atatcttttc 180

catcttcatc caccattact cttcttatgg cacttgatat ctcatctctt tgaaacccat 240

tctctaaata cacccccacc ttcgagacat cgctcatgta tcgcgcgtca atcggttgat 300

cgccccaaaa aggcgaacaa atcattgcaa caccttcaca aacactctcc aaagtcgagt 360

tccatccgct atgagtccaa aacgcacccg ttgcttcatg agctaaaact tcttgttgag 420

gagcccattt aacaactcgc cccctttcac ctgggaaccc atctggcaat ggcaatgact 480

cgagccatgt tgaatcttta acgaacccta gtcgaaccac ccataaaaag ggttgttggc 540

tatagaccaa cccgtgagct atttccaaga actctttctc atccacttga caaaggctac 600

caaaactaac atagagtaca gacttgggtg cttgttggtc taaccaagga aaaatggttc 660

gatcttgttc tagtaaacta ctcgatgaag cgttaaaata cttgtggaat ggtataatga 720

aacgtggtat cgggaaatca tgtggaatgc tatcgagctc ggtttcttcg agttctttga 780

aagagttcca aatgattccc gatgatgctt ttatttgttt aaccatttcg cgtatcatct 840

tcgcatatgg gtcattcatg ctctttattc caatcttttt tatgtctttc acttttagta 900

atggaaactc cattacttgt tcttccaaat gactgtcatc aagtgcgaag taaccacgat 960

catcaaaaag gggaatggaa gcataaacaa tagaacaata aacgctagtt gtcctcaaaa 1020

caattctcgg aatctgaaga ctatcagcca ccgattgcgt gaagtgccaa atcgcgtcag 1080

tgatcaaaca cgataccggc tcatcgtttc cagaagctaa caataattcc agtttatgac 1140

gtaattcatc tgcaccgtct tgtttaaagg tataattttg agcgaattcg aacgacgatg 1200

atgtggaaaa cggttcgttt tcggcgttat cgagaactga tatgaaagtg aagttagggt 1260

aattagagaa tttgggcgcg ttgaagtttg tgtgaaggat gacgatgctg aatccattgg 1320

aatagagaag attggctagc tgaatcattg ggtttatatg accttgaaat ggcatcggaa 1380

aaagtattat tctccggtgc cgacgaacgg ttgtttgtgg ttggttctcc at 1432

<210> 2

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<212> PRT

<213> Stevia rebaudiana (Bertoni) Hemsl)

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Met Glu Asn Gln Pro Gln Thr Thr Val Arg Arg His Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Met Pro Phe Gln Gly His Ile Asn Pro Met Ile Gln Leu

20 25 30

Ala Asn Leu Leu Tyr Ser Asn Gly Phe Ser Ile Val Ile Leu His Thr

35 40 45

Asn Phe Asn Ala Pro Lys Phe Ser Asn Tyr Pro Asn Phe Thr Phe Ile

50 55 60

Ser Val Leu Asp Asn Ala Glu Asn Glu Pro Phe Ser Thr Ser Ser Ser

65 70 75 80

Phe Glu Phe Ala Gln Asn Tyr Thr Phe Lys Gln Asp Gly Ala Asp Glu

85 90 95

Leu Arg His Lys Leu Glu Leu Leu Leu Ala Ser Gly Asn Asp Glu Pro

100 105 110

Val Ser Cys Leu Ile Thr Asp Ala Ile Trp His Phe Thr Gln Ser Val

115 120 125

Ala Asp Ser Leu Gln Ile Pro Arg Ile Val Leu Arg Thr Thr Ser Val

130 135 140

Tyr Cys Ser Ile Val Tyr Ala Ser Ile Pro Leu Phe Asp Asp Arg Gly

145 150 155 160

Tyr Phe Ala Leu Asp Asp Ser His Leu Glu Glu Gln Val Met Glu Phe

165 170 175

Pro Leu Leu Lys Val Lys Asp Ile Lys Lys Ile Gly Ile Lys Ser Met

180 185 190

Asn Asp Pro Tyr Ala Lys Met Ile Arg Glu Met Val Lys Gln Ile Lys

195 200 205

Ala Ser Ser Gly Ile Ile Trp Asn Ser Phe Lys Glu Leu Glu Glu Thr

210 215 220

Glu Leu Asp Ser Ile Pro His Asp Phe Pro Ile Pro Arg Phe Ile Ile

225 230 235 240

Pro Phe His Lys Tyr Phe Asn Ala Ser Ser Ser Ser Leu Leu Glu Gln

245 250 255

Asp Arg Thr Ile Phe Pro Trp Leu Asp Gln Gln Ala Pro Lys Ser Val

260 265 270

Leu Tyr Val Ser Phe Gly Ser Leu Cys Gln Val Asp Glu Lys Glu Phe

275 280 285

Leu Glu Ile Ala His Gly Leu Val Tyr Ser Gln Gln Pro Phe Leu Trp

290 295 300

Val Val Arg Leu Gly Phe Val Lys Asp Ser Thr Trp Leu Glu Ser Leu

305 310 315 320

Pro Leu Pro Asp Gly Phe Pro Gly Glu Arg Gly Arg Val Val Lys Trp

325 330 335

Ala Pro Gln Gln Glu Val Leu Ala His Glu Ala Thr Gly Ala Phe Trp

340 345 350

Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu Gly Val

355 360 365

Ala Met Ile Cys Ser Pro Phe Trp Gly Asp Gln Pro Ile Asp Ala Arg

370 375 380

Tyr Met Ser Asp Val Ser Lys Val Gly Val Tyr Leu Glu Asn Gly Phe

385 390 395 400

Gln Arg Asp Glu Ile Ser Ser Ala Ile Arg Arg Val Met Val Asp Glu

405 410 415

Asp Gly Lys Asp Ile Arg Glu Arg Val Lys Val Leu Lys Gln Lys Val

420 425 430

Asp Asp Ser Leu Met Ile Ser Gly Ser Ser Asn Glu Ser Leu Glu Ser

435 440 445

Leu Ile His Tyr Ile Ser Ser Phe

450 455

<210> 3

<211> 1576

<212> DNA

<213> Stevia rebaudiana (Bertoni) Hemsl)

<400> 3

atggcttcaa tagcagaaat gcaaaagcca catgccatat gcatccccta cccagcccaa 60

ggccacatca accccatgat gcaatttgct aagctccttc acttcaaagg ctttcacata 120

tcttttgtca ataaccacta caaccataag cggttgcagc ggtcccgcgg tctgtccgcc 180

ctcgaaggtc tacctgattt tcatttctac tcgattcccg atggccttcc gccttcaaat 240

gctgaggcca cccagtcgat ccccgggcta tgtgagtcga ttcctaagca cagtttggaa 300

ccattttgtg aattgatcgc tacgctaaat ggttcggacg tgccacctgt aagctgtata 360

atctctgatg gggtcatgag ctttacgctt caagctgccg agaggttcgg gttgccggaa 420

gttttgttct ggaccccaag tgcttgtggg tttttggctt acactcacta tcgagatctt 480

gtggataagg agtatattcc cctcaaagac acgaacgact tgacaaatgg gtatttagaa 540

acaagcttgg attggattcc tgggatgaaa aacatccgat taaaagattt cccatccttt 600

attcgaacca cagacataaa tgatattatg ctcaattatt tcttgattga aaccgaagcc 660

atcccaaaag gcgtagcgat cattcttaac acatttgacg cgttagaaaa agatagtatt 720

acgcctgtac ttgctctaaa tccacaaata tacaccattg gtccattaca catgatgcaa 780

caatatgtcg atcatgatga gagactcaaa cacattgggt ccaacctttg gaaggaagat 840

gtgagctgca tcaattggct tgacaccaaa aagcctaatt cggttgttta tgtgaacttt 900

ggaagtatta cggttatgac gaaagaacaa ctgatcgagt ttgggtgggg actggctaat 960

agcaagaagg atttcttgtg gataacgagg cctgatattg ttggaggcaa tgaagccatg 1020

ataccaccag agttcataga ggagaccaaa gaaaggggca tggttactag ctggtgctct 1080

caggaagagg ttttaaaaca tccatcaatc ggggtattct tgactcatag tggatggaac 1140

tcaaccattg agagtattag caacggtgtt cccatgattt gttggccttt tttgcagagc 1200

aacaaacaaa ttgtcggtat tgttgtgttg aatgggaaat tggattggaa attgatacag 1260

atgtgaagag agaggaggta gaggctcaag tgagggagat gatggatggg tcgaaaggga 1320

agatgatgaa aaacaaggct ttggaatgga agaagaactc tgaagaagcg gtatccattg 1380

gtggatcatc ttatctcaac tttgaaaaat tagttaccga tgttctttta agaaagggat 1440

cctgccccga gcgagctata tgaagttttc agtgtgccac tgattcggag taccctttaa 1500

atatgactat ttctattttg cctctgaaca ttctgatcgt tgaagatcat gccttgctag 1560

ccgatggcat ccgtaa 1576

<210> 4

<211> 421

<212> PRT

<213> Stevia rebaudiana (Bertoni) Hemsl)

<400> 4

Met Ala Ser Ile Ala Glu Met Gln Lys Pro His Ala Ile Cys Ile Pro

1 5 10 15

Tyr Pro Ala Gln Gly His Ile Asn Pro Met Met Gln Phe Ala Lys Leu

20 25 30

Leu His Phe Lys Gly Phe His Ile Ser Phe Val Asn Asn His Tyr Asn

35 40 45

His Lys Arg Leu Gln Arg Ser Arg Gly Leu Ser Ala Leu Glu Gly Leu

50 55 60

Pro Asp Phe His Phe Tyr Ser Ile Pro Asp Gly Leu Pro Pro Ser Asn

65 70 75 80

Ala Glu Ala Thr Gln Ser Ile Pro Gly Leu Cys Glu Ser Ile Pro Lys

85 90 95

His Ser Leu Glu Pro Phe Cys Glu Leu Ile Ala Thr Leu Asn Gly Ser

100 105 110

Asp Val Pro Pro Val Ser Cys Ile Ile Ser Asp Gly Val Met Ser Phe

115 120 125

Thr Leu Gln Ala Ala Glu Arg Phe Gly Leu Pro Glu Val Leu Phe Trp

130 135 140

Thr Pro Ser Ala Cys Gly Phe Leu Ala Tyr Thr His Tyr Arg Asp Leu

145 150 155 160

Val Asp Lys Glu Tyr Ile Pro Leu Lys Asp Thr Asn Asp Leu Thr Asn

165 170 175

Gly Tyr Leu Glu Thr Ser Leu Asp Trp Ile Pro Gly Met Lys Asn Ile

180 185 190

Arg Leu Lys Asp Phe Pro Ser Phe Ile Arg Thr Thr Asp Ile Asn Asp

195 200 205

Ile Met Leu Asn Tyr Phe Leu Ile Glu Thr Glu Ala Ile Pro Lys Gly

210 215 220

Val Ala Ile Ile Leu Asn Thr Phe Asp Ala Leu Glu Lys Asp Ser Ile

225 230 235 240

Thr Pro Val Leu Ala Leu Asn Pro Gln Ile Tyr Thr Ile Gly Pro Leu

245 250 255

His Met Met Gln Gln Tyr Val Asp His Asp Glu Arg Leu Lys His Ile

260 265 270

Gly Ser Asn Leu Trp Lys Glu Asp Val Ser Cys Ile Asn Trp Leu Asp

275 280 285

Thr Lys Lys Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser Ile Thr

290 295 300

Val Met Thr Lys Glu Gln Leu Ile Glu Phe Gly Trp Gly Leu Ala Asn

305 310 315 320

Ser Lys Lys Asp Phe Leu Trp Ile Thr Arg Pro Asp Ile Val Gly Gly

325 330 335

Asn Glu Ala Met Ile Pro Pro Glu Phe Ile Glu Glu Thr Lys Glu Arg

340 345 350

Gly Met Val Thr Ser Trp Cys Ser Gln Glu Glu Val Leu Lys His Pro

355 360 365

Ser Ile Gly Val Phe Leu Thr His Ser Gly Trp Asn Ser Thr Ile Glu

370 375 380

Ser Ile Ser Asn Gly Val Pro Met Ile Cys Trp Pro Phe Leu Gln Ser

385 390 395 400

Asn Lys Gln Ile Val Gly Ile Val Val Leu Asn Gly Lys Leu Asp Trp

405 410 415

Lys Leu Ile Gln Met

420

<210> 5

<211> 1476

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 5

atggcatcgg aatttcgtcc tcctcttcat tttgttctct tccctttcat ggctcaaggc 60

cacatgatcc caatggtaga tattgcaagg ctcctggctc agcgcggggt gactataacc 120

attgtcacta cacctcaaaa cgcaggccgg ttcaagaacg ttcttagccg ggctatccaa 180

tccggcttgc ccatcaatct cgtgcaagta aagtttccat ctcaagaatc gggttcaccg 240

gaaggacagg agaatttgga cttgctcgat tcattggggg cttcattaac cttcttcaaa 300

gcatttagcc tgctcgagga accagtcgag aagctcttga aagagattca acctaggcca 360

aactgcataa tcgctgacat gtgtttgcct tatacaaaca gaattgccaa gaatcttggt 420

ataccaaaaa tcatctttca tggcatgtgt tgcttcaatc ttctttgtac gcacataatg 480

caccaaaacc acgagttctt ggaaactata gagtctgaca aggaatactt ccccattcct 540

aatttccctg acagagttga gttcacaaaa tctcagcttc caatggtatt agttgctgga 600

gattggaaag acttccttga cggaatgaca gaaggggata acacttctta tggtgtgatt 660

gttaacacgt ttgaagagct cgagccagct tatgttagag actacaagaa ggttaaagcg 720

ggtaagatat ggagcatcgg accggtttcc ttgtgcaaca agttaggaga agaccaagct 780

gagaggggaa acaaggcgga cattgatcaa gacgagtgta ttaaatggct tgattctaaa 840

gaagaagggt cggtgctata tgtttgcctt ggaagtatat gcaatcttcc tctgtctcag 900

ctcaaagagc tcggcttagg cctcgaggaa tcccaaagac ctttcatttg ggtcataaga 960

ggttgggaga agtataacga gttacttgaa tggatctcag agagcggtta taaggaaaga 1020

atcaaagaaa gaggccttct cataacagga tggtcgcctc aaatgcttat ccttacacat 1080

cctgccgttg gaggattctt gacacattgt ggatggaact ctactcttga aggaatcact 1140

tcaggcgttc cattactcac gtggccactg tttggagacc aattctgcaa tgagaaattg 1200

gcggtgcaga tactaaaagc cggtgtgaga gctggggttg aagagtccat gagatgggga 1260

gaagaggaga aaataggagt actggtggat aaagaaggag taaagaaggc agtggaggaa 1320

ttgatgggtg atagtaatga tgctaaggag agaagaaaaa gagtgaaaga gcttggagaa 1380

ttagctcaca aggctgtgga agaaggaggc tcttctcatt ccaacatcac attcttgcta 1440

caagacataa tgcaattaga acaacccaag aaatga 1476

<210> 6

<211> 491

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 6

Met Ala Ser Glu Phe Arg Pro Pro Leu His Phe Val Leu Phe Pro Phe

1 5 10 15

Met Ala Gln Gly His Met Ile Pro Met Val Asp Ile Ala Arg Leu Leu

20 25 30

Ala Gln Arg Gly Val Thr Ile Thr Ile Val Thr Thr Pro Gln Asn Ala

35 40 45

Gly Arg Phe Lys Asn Val Leu Ser Arg Ala Ile Gln Ser Gly Leu Pro

50 55 60

Ile Asn Leu Val Gln Val Lys Phe Pro Ser Gln Glu Ser Gly Ser Pro

65 70 75 80

Glu Gly Gln Glu Asn Leu Asp Leu Leu Asp Ser Leu Gly Ala Ser Leu

85 90 95

Thr Phe Phe Lys Ala Phe Ser Leu Leu Glu Glu Pro Val Glu Lys Leu

100 105 110

Leu Lys Glu Ile Gln Pro Arg Pro Asn Cys Ile Ile Ala Asp Met Cys

115 120 125

Leu Pro Tyr Thr Asn Arg Ile Ala Lys Asn Leu Gly Ile Pro Lys Ile

130 135 140

Ile Phe His Gly Met Cys Cys Phe Asn Leu Leu Cys Thr His Ile Met

145 150 155 160

His Gln Asn His Glu Phe Leu Glu Thr Ile Glu Ser Asp Lys Glu Tyr

165 170 175

Phe Pro Ile Pro Asn Phe Pro Asp Arg Val Glu Phe Thr Lys Ser Gln

180 185 190

Leu Pro Met Val Leu Val Ala Gly Asp Trp Lys Asp Phe Leu Asp Gly

195 200 205

Met Thr Glu Gly Asp Asn Thr Ser Tyr Gly Val Ile Val Asn Thr Phe

210 215 220

Glu Glu Leu Glu Pro Ala Tyr Val Arg Asp Tyr Lys Lys Val Lys Ala

225 230 235 240

Gly Lys Ile Trp Ser Ile Gly Pro Val Ser Leu Cys Asn Lys Leu Gly

245 250 255

Glu Asp Gln Ala Glu Arg Gly Asn Lys Ala Asp Ile Asp Gln Asp Glu

260 265 270

Cys Ile Lys Trp Leu Asp Ser Lys Glu Glu Gly Ser Val Leu Tyr Val

275 280 285

Cys Leu Gly Ser Ile Cys Asn Leu Pro Leu Ser Gln Leu Lys Glu Leu

290 295 300

Gly Leu Gly Leu Glu Glu Ser Gln Arg Pro Phe Ile Trp Val Ile Arg

305 310 315 320

Gly Trp Glu Lys Tyr Asn Glu Leu Leu Glu Trp Ile Ser Glu Ser Gly

325 330 335

Tyr Lys Glu Arg Ile Lys Glu Arg Gly Leu Leu Ile Thr Gly Trp Ser

340 345 350

Pro Gln Met Leu Ile Leu Thr His Pro Ala Val Gly Gly Phe Leu Thr

355 360 365

His Cys Gly Trp Asn Ser Thr Leu Glu Gly Ile Thr Ser Gly Val Pro

370 375 380

Leu Leu Thr Trp Pro Leu Phe Gly Asp Gln Phe Cys Asn Glu Lys Leu

385 390 395 400

Ala Val Gln Ile Leu Lys Ala Gly Val Arg Ala Gly Val Glu Glu Ser

405 410 415

Met Arg Trp Gly Glu Glu Glu Lys Ile Gly Val Leu Val Asp Lys Glu

420 425 430

Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Asp Ser Asn Asp Ala

435 440 445

Lys Glu Arg Arg Lys Arg Val Lys Glu Leu Gly Glu Leu Ala His Lys

450 455 460

Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn Ile Thr Phe Leu Leu

465 470 475 480

Gln Asp Ile Met Gln Leu Glu Gln Pro Lys Lys

485 490

<210> 7

<211> 1488

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 7

atggtttccg aaacaaccaa atcttctcca cttcactttg ttctcttccc tttcatggct 60

caaggccaca tgattcccat ggttgatatt gcaaggctct tggctcagcg tggtgtgatc 120

ataacaattg tcacgacgcc tcacaatgca gcgaggttca agaatgtcct aaaccgtgcc 180

attgagtctg gcttgcccat caacttagtg caagtcaagt ttccatatct agaagctggt 240

ttgcaagaag gacaagagaa tatcgattct cttgacacaa tggagcggat gatacctttc 300

tttaaagcgg ttaactttct cgaagaacca gtccagaagc tcattgaaga gatgaaccct 360

cgaccaagct gtctaatttc tgatttttgt ttgccttata caagcaaaat cgccaagaag 420

ttcaatatcc caaagatcct cttccatggc atgggttgct tttgtcttct gtgtatgcat 480

gttttacgca agaaccgtga gatcttggac aatttaaagt cagataagga gcttttcact 540

gttcctgatt ttcctgatag agttgaattc acaagaacgc aagttccggt agaaacatat 600

gttccagctg gagactggaa agatatcttt gatggtatgg tagaagcgaa tgagacatct 660

tatggtgtga tcgtcaactc atttcaagag ctcgagcctg cttatgccaa agactacaag 720

gaggtaaggt ccggtaaagc atggaccatt ggacccgttt ccttgtgcaa caaggtagga 780

gccgacaaag cagagagggg aaacaaatca gacattgatc aagatgagtg ccttaaatgg 840

ctcgattcta agaaacatgg ctcggtgctt tacgtttgtc ttggaagtat ctgtaatctt 900

cctttgtctc aactcaagga gctgggacta ggcctagagg aatcccaaag acctttcatt 960

tgggtcataa gaggttggga gaagtacaaa gagttagttg agtggttctc ggaaagcggc 1020

tttgaagata gaatccaaga tagaggactt ctcatcaaag gatggtcccc tcaaatgctt 1080

atcctttcac atccatcagt tggagggttc ctaacacact gtggttggaa ctcgactctt 1140

gaggggataa ctgctggtct accgctactt acatggccgc tattcgcaga ccaattctgc 1200

aatgagaaat tggtcgttga ggtactaaaa gccggtgtaa gatccggggt tgaacagcct 1260

atgaaatggg gagaagagga gaaaatagga gtgttggtgg ataaagaagg agtgaagaag 1320

gcagtggaag aattaatggg tgagagtgat gatgcaaaag agagaagaag aagagccaaa 1380

gagcttggag attcagctca caaggctgtg gaagaaggag gctcttctca ttctaacatc 1440

tctttcttgc tacaagacat aatggaactg gcagaaccca ataattga 1488

<210> 8

<211> 495

<212> PRT

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 8

Met Val Ser Glu Thr Thr Lys Ser Ser Pro Leu His Phe Val Leu Phe

1 5 10 15

Pro Phe Met Ala Gln Gly His Met Ile Pro Met Val Asp Ile Ala Arg

20 25 30

Leu Leu Ala Gln Arg Gly Val Ile Ile Thr Ile Val Thr Thr Pro His

35 40 45

Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala Ile Glu Ser Gly

50 55 60

Leu Pro Ile Asn Leu Val Gln Val Lys Phe Pro Tyr Leu Glu Ala Gly

65 70 75 80

Leu Gln Glu Gly Gln Glu Asn Ile Asp Ser Leu Asp Thr Met Glu Arg

85 90 95

Met Ile Pro Phe Phe Lys Ala Val Asn Phe Leu Glu Glu Pro Val Gln

100 105 110

Lys Leu Ile Glu Glu Met Asn Pro Arg Pro Ser Cys Leu Ile Ser Asp

115 120 125

Phe Cys Leu Pro Tyr Thr Ser Lys Ile Ala Lys Lys Phe Asn Ile Pro

130 135 140

Lys Ile Leu Phe His Gly Met Gly Cys Phe Cys Leu Leu Cys Met His

145 150 155 160

Val Leu Arg Lys Asn Arg Glu Ile Leu Asp Asn Leu Lys Ser Asp Lys

165 170 175

Glu Leu Phe Thr Val Pro Asp Phe Pro Asp Arg Val Glu Phe Thr Arg

180 185 190

Thr Gln Val Pro Val Glu Thr Tyr Val Pro Ala Gly Asp Trp Lys Asp

195 200 205

Ile Phe Asp Gly Met Val Glu Ala Asn Glu Thr Ser Tyr Gly Val Ile

210 215 220

Val Asn Ser Phe Gln Glu Leu Glu Pro Ala Tyr Ala Lys Asp Tyr Lys

225 230 235 240

Glu Val Arg Ser Gly Lys Ala Trp Thr Ile Gly Pro Val Ser Leu Cys

245 250 255

Asn Lys Val Gly Ala Asp Lys Ala Glu Arg Gly Asn Lys Ser Asp Ile

260 265 270

Asp Gln Asp Glu Cys Leu Lys Trp Leu Asp Ser Lys Lys His Gly Ser

275 280 285

Val Leu Tyr Val Cys Leu Gly Ser Ile Cys Asn Leu Pro Leu Ser Gln

290 295 300

Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Gln Arg Pro Phe Ile

305 310 315 320

Trp Val Ile Arg Gly Trp Glu Lys Tyr Lys Glu Leu Val Glu Trp Phe

325 330 335

Ser Glu Ser Gly Phe Glu Asp Arg Ile Gln Asp Arg Gly Leu Leu Ile

340 345 350

Lys Gly Trp Ser Pro Gln Met Leu Ile Leu Ser His Pro Ser Val Gly

355 360 365

Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly Ile Thr

370 375 380

Ala Gly Leu Pro Leu Leu Thr Trp Pro Leu Phe Ala Asp Gln Phe Cys

385 390 395 400

Asn Glu Lys Leu Val Val Glu Val Leu Lys Ala Gly Val Arg Ser Gly

405 410 415

Val Glu Gln Pro Met Lys Trp Gly Glu Glu Glu Lys Ile Gly Val Leu

420 425 430

Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu

435 440 445

Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Ala Lys Glu Leu Gly Asp

450 455 460

Ser Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn Ile

465 470 475 480

Ser Phe Leu Leu Gln Asp Ile Met Glu Leu Ala Glu Pro Asn Asn

485 490 495

<210> 9

<211> 1377

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 9

atggaaaata aaacggagac caccgttcgc cggcgccgga gaataatatt attcccggta 60

ccatttcaag gccacattaa cccaattctt cagctagcca atgtgttgta ctctaaagga 120

ttcagtatca ccatctttca caccaacttc aacaaaccca aaacatctaa ttaccctcac 180

ttcactttca gattcatcct cgacaacgac ccacaagacg aacgcatttc caatctaccg 240

actcatggtc cgctcgctgg tatgcggatt ccgattatca acgaacacgg agctgacgaa 300

ttacgacgcg aactggaact gttgatgtta gcttctgaag aagatgaaga ggtatcgtgt 360

ttaatcacgg atgctctttg gtacttcgcg caatctgttg ctgacagtct taacctccga 420

cggcttgttt tgatgacaag cagcttgttt aattttcatg cacatgtttc acttcctcag 480

tttgatgagc ttggttacct cgatcctgat gacaaaaccc gtttggaaga acaagcgagt 540

gggtttccta tgctaaaagt gaaagacatc aagtctgcgt attcgaactg gcaaatactc 600

aaagagatat tagggaagat gataaaacaa acaaaagcat cttcaggagt catctggaac 660

tcatttaagg aactcgaaga gtctgagctc gaaactgtta tccgtgagat cccggctcca 720

agtttcttga taccactccc caagcatttg acagcctctt ccagcagctt actagaccac 780

gatcgaaccg tttttcaatg gttagaccaa caaccgccaa gttcggtact gtatgttagt 840

tttggtagta ctagtgaagt ggatgagaaa gatttcttgg aaatagctcg tgggttggtt 900

gatagcaagc agtcgttttt atgggtggtt cgacctgggt ttgtcaaggg ttcgacgtgg 960

gtcgaaccgt tgccagatgg gttcttgggt gaaagaggac gtattgtgaa atgggttcca 1020

cagcaagaag tgctagctca tggagcaata ggcgcattct ggactcatag cggatggaac 1080

tctacgttgg aaagcgtttg tgaaggtgtt cctatgattt tctcggattt tgggctcgat 1140

caaccgttga atgctagata catgagtgat gttttgaagg taggggtgta tttggaaaat 1200

gggtgggaaa gaggagagat agcaaatgca ataagaagag ttatggtgga tgaagaagga 1260

gaatacatta gacagaatgc aagagttttg aaacaaaagg cagatgtttc tttgatgaag 1320

ggtggttcgt cttacgaatc attagagtct ctagtttctt acatttcatc gttgtaa 1377

<210> 10

<211> 458

<212> PRT

<213> Stevia rebaudiana (Bertoni) Hemsl)

<400> 10

Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu

20 25 30

Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr

35 40 45

Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg

50 55 60

Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro

65 70 75 80

Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His

85 90 95

Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser

100 105 110

Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr

115 120 125

Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu

130 135 140

Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln

145 150 155 160

Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu

165 170 175

Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser

180 185 190

Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile

195 200 205

Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu

210 215 220

Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu

450 455

<210> 11

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

agagagccat ggatggcttc aatagcagaa atg 33

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

agagagggat ccctttctta aaagaacatc gg 32

<210> 13

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

cgggatccat ggcatcggaa tttcgtc 27

<210> 14

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

aggcggccgc tcatttcttg ggttgttc 28

<210> 15

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

cgggatccat ggtttccgaa acaacc 26

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

aggcggccgc tcaattattg ggttctgcc 29

<210> 17

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

cgcgcgggat ccatggagaa ccaaccacaa ac 32

<210> 18

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

aggcggccgc ttagaatgac gaaatataat g 31

<210> 19

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

agagagggat ccatggaaaa taaaacggag ac 32

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

aggcggccgc ttacaacgat gaaatgtaag 30

Claims

1. A polypeptide, wherein the polypeptide is shown as SEQ ID NO. 2.

2. A polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence encoding the polypeptide of claim 1; and

(2) a polynucleotide sequence which is fully complementary to the polynucleotide sequence of (1).

3. The polynucleotide sequence of claim 2, wherein the polynucleotide sequence is selected from the group consisting of:

(a) 1, or a polynucleotide sequence shown in SEQ ID NO; and

(b) 1, the complete complement of SEQ ID NO.

4. A nucleic acid construct comprising the polynucleotide sequence of claim 2 or 3.

5. The nucleic acid construct of claim 4, wherein said nucleic acid construct is a cloning vector or an expression vector.

6. A genetically engineered host cell, wherein the host cell:

(1) expressing the polypeptide of claim 1; and/or

(2) Comprising the nucleic acid construct of claim 4.

7. The host cell of claim 6, wherein the host cell is Agrobacterium.

8. The host cell of claim 6 or 7, wherein the host cell further:

(a) expressing one or more of SEQ ID NO 4, 6, 8, 10, and/or

(b) Contains one or more expression vectors expressing SEQ ID NO 4, 6, 8 and 10.

9. The host cell of claim 8,

the host cell expresses at least SEQ ID NO 4, 6 and 8, and/or

The host cell contains at least expression vectors for expression of SEQ ID NO 4, 6 and 8.

10. The application of (a) or (a) and (b) in preparing stevioside as follows:

(a) the polypeptide, its coding sequence or expression vector of claim 1,

(b) one or more of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10, a coding sequence or an expression vector thereof.

11. Use of a genetically engineered host cell expressing (a), or (a) and (b), as described below, in the preparation of stevioside:

(a) the polypeptide of claim 1, wherein said polypeptide is,

(b) one or more of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10.

12. Use of the polypeptide of claim 1, a coding sequence or an expression vector thereof, or a genetically engineered host cell expressing the polypeptide of claim 1, for the synthesis of rebaudioside a.

13. A method of constructing a transgenic plant, the method comprising:

(1) providing an agrobacterium carrying an expression vector expressing the polypeptide of claim 1;

(3) selecting a plant cell or tissue transformed with the coding sequence; and

(4) regenerating the plant cell or tissue of step (3) into a plant.

14. The method of claim 13, wherein the agrobacterium further carries an expression vector expressing one or more of SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, and SEQ ID No. 10.