CN108359652A - Glycosyl transferase and its application - Google Patents

Glycosyl transferase and its application Download PDF

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CN108359652A
CN108359652A CN201710061097.0A CN201710061097A CN108359652A CN 108359652 A CN108359652 A CN 108359652A CN 201710061097 A CN201710061097 A CN 201710061097A CN 108359652 A CN108359652 A CN 108359652A
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glycosyl transferase
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CN108359652B (en
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肖友利
尤文静
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Center for Excellence in Molecular Plant Sciences of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Abstract

Glycosyl transferase of the present invention and its application.Specifically, the present invention provides a kind of polypeptide, the polypeptide is selected from：(1)SEQ ID NO:Polypeptide shown in 2；(2) by SEQ ID NO:Amino acid sequence shown in 2 and promotion SEQ ID NO:The polypeptide of the sequence composition of expression, secretion and/or the purifying of amino acid sequence shown in 2.The invention further relates to relevant polynucleotide sequence, nucleic acid constructs, genetically engineered host cell and applications.The further perfect biosynthesis pathway of steviol glycoside of the present invention；Using synthetic biology, it is used to improve the industrialization of stevioside content by transgenic technology.

Description

Glycosyl transferase and its application
Technical field
The present invention relates to glycosyl transferase and its applications.
Background technology
STEVIA REBAUDIANA originates in South America Paraguay, is a kind of composite family herbaceos perennial.Stevioside is from stevioside leaf The highest component of content is extracted in piece, it is the general name of a variety of steviosides.Stevioside accounts for all sugar in STEVIA REBAUDIANA blade 60%-70%, stevioside (stevioside) and rebaudioside (Rebaudioside) A are most important in STEVIA REBAUDIANA blade Ingredient.The sugariness of stevioside is far more than the sugariness of sucrose, and as a kind of low heat value sweetener, it is optimal cane sugar substitution Product.Stevioside is also applied to pharmaceuticals industry other than being applied to food industry as sweetener, has blood pressure lowering and hypoglycemic Effect also has hyperlipemic patients fine curative effect.
The method of industrialized production stevioside is refined after being extracted in STEVIA REBAUDIANA blade mostly, the extraction of stevioside It is divided into two kinds of water extraction method and organic solvent extraction.
Recent years, as the continuous development of this emerging field of synthetic biology will synthesize sweet tea by transgenic technology Related gene in chrysanthemum glycosides metabolic pathway is assembled, and builds the engineered strain of the biosynthesis pathway containing stevioside to improve stevia rebaudianum Glycosides contains quantifier elimination and industrialization, has preferable facilitation to improving stevioside yield.
Invention content
First aspect present invention provides polypeptide, and the polypeptide is selected from：
(1)SEQ ID NO:Polypeptide shown in 2；With
(2) by SEQ ID NO:Amino acid sequence shown in 2 and promotion SEQ ID NO:The table of amino acid sequence shown in 2 The polypeptide for amino acid or the amino acid sequence composition for reaching, secreting and/or purifying.
Second aspect of the present invention provides polynucleotide sequence, and the polynucleotide sequence is selected from：
(1) polynucleotide sequence of polypeptide described in first aspect present invention is encoded；
(2) with the polynucleotide sequence of (1) described polynucleotide sequence complementation；With
(3) segment of the long 10-40 base of (1) or (2) described polynucleotide sequence.
In one or more embodiments, the polynucleotide sequence is selected from：
(a)SEQ ID NO:Polynucleotide sequence shown in 1；
(b)SEQ ID NO:1 complementary series；With
(c)SEQ ID NO:1 or its complementary series long 10-40 base segment.
Third aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains described in second aspect of the present invention Polynucleotide sequence.
In one or more embodiments, the nucleic acid constructs is cloning vector or expression vector.
Fourth aspect present invention provides a kind of genetically engineered host cell, the host cell：
(1) polypeptide described in first aspect present invention is expressed；And/or
(2) containing the nucleic acid constructs described in third aspect present invention.
In one or more embodiments, the host cell also (a) expresses SEQ ID NO:4, one in 6,8 and 10 Kind or multiple polypeptides, and/or (b) contain expression SEQ ID NO:4, one or more in 6,8 and 10 expression vector.
Fifth aspect present invention provides a kind of method synthesizing steviol -19-O- steviosides or sweetness agent classification glycosides, described Method includes making SEQ ID NO:4, the step of one or more polypeptide glycosylation substrate steviols in 6,8 and 10.
Sixth aspect present invention provides a kind of method of synthesis steviol -13-O- steviosides, and the method includes using SEQ ID NO:The step of polypeptide glycosylation substrate steviol shown in 6 and/or 8.
Seventh aspect present invention provides a kind of method of synthesis content rebaudioside-A, and the method includes using the present invention first The step of aspect polypeptide glycosylation substrate stevioside.
Eighth aspect present invention provides a kind of method of structure genetically modified plants, the method includes：
(1) Agrobacterium for carrying expression vector is provided, the expression vector expresses polypeptide of the present invention；
(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), the coded sequence is made to be transferred to plant Cell, and be integrated on the chromosome of plant cell；
(3) plant cell or tissue for being transferred to the coded sequence are selected；With
(4) by step (3) plant cell or regeneration at plant.
Ninth aspect present invention provides the compound X that structure is shown below：
In one or more embodiments, the compound X is used as sweetener.
Tenth aspect present invention provides a kind of method preparing above compound X, and the method includes using SEQ ID NO:10 or its remain glycosyl transferase activity mutant glycosylation rebaudioside I the step of.
The present invention also provides SEQ ID NO:2 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:4 or It remains the mutant of glycosyl transferase activity, SEQ ID NO:6 or its remain the mutant SEQ of glycosyl transferase activity ID NO:8 or its remain the mutant of glycosyl transferase activity and SEQ ID NO:10 or its remain glycosyl transferase work One or more in the mutant of property, coded sequence, expression vector or genetically engineered host cell of the present invention exist The application in stevioside, or the application in preparing stevioside, or the application in preparing content rebaudioside-A are prepared, or is being made Application in standby compound X.
In one or more embodiments, the host cell expression SEQ ID NO:2 or its remain glycosyl transfer Mutant, the SEQ ID NO of enzymatic activity:4 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:6 or its guarantor Mutant, the SEQ ID NO of glycosyl transferase activity are stayed:8 or its remain the mutant of glycosyl transferase activity and SEQ ID NO:10 or its remain it is one or more in the mutant of glycosyl transferase activity.
In one or more embodiments, the genetically engineered host cell at least expresses SEQ ID NO:2 or It remains the mutant of glycosyl transferase activity, while also expression SEQ ID NO:4,6 and 8 or they remain glycosyl transfer The mutant of enzymatic activity.In certain embodiments, the host cell further also expresses SEQ ID NO:10 or its reservation The mutant of glycosyl transferase activity.In one or more embodiments, the host cell also expresses stevioside conjunction At other glycosyl transferases in approach.
The present invention also provides SEQ ID NO:2 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:4 or It remains the mutant of glycosyl transferase activity, SEQ ID NO:6 or its remain the mutant SEQ of glycosyl transferase activity ID NO:8 or its remain the mutant of glycosyl transferase activity and SEQ ID NO:10 or its remain glycosyl transferase work One or more in the mutant of property, coded sequence, expression vector or genetically engineered host cell of the present invention exist Prepare the application in following formula: compound：
In formula,
R1It is selected from：H、Glcβ1-、Glcβ1-3Glcβ1-、Glcβ1-2Glcβ1-、Glcβ1-2(Glcβ1-3)-Glcβ1-、 Glc β 1-2 (Glc β 1-6)-Glc β 1- and Glc β 1-3 (Glc β 1-6)-Glc β 1-；With
R2It is selected 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- steviosides, steviol - 19-O- steviosides, sweetness agent classification glycosides, steviolbioside, stevioside, content rebaudioside-A, rebaudioside I, rebaudioside D, Lay Bao Di glucosides M, rebaudioside M2 and compound X.
Description of the drawings
Fig. 1：Using cDNA as the electrophoresis result of template amplification glycosyl transferase related gene segment.
Fig. 2：The electrophoresis detection of the SDS-PAGE of SrUGT85A8.M：Albumen Marker, CL：Cell pyrolysis liquid, CP：It is broken Cell precipitation, CS：Smudge cells supernatant, FT：Supernatant, WB：It is eluted without imidazole buffer and is not associated with albumen, 1-6：15mM, 25mM, 50mM, 100mM, 250mM, 250mM imidazoles elute.
Fig. 3：The electrophoresis detection of the SDS-PAGE of SrUGT76G2.M：Albumen Marker, CL：Cell pyrolysis liquid, CS：It is broken Cell conditioned medium, CP：Smudge cells precipitate, FT：Supernatant, WB：It is eluted without imidazole buffer and is not associated with albumen, 1-4：15mM, 50mM, 100mM, 250mM imidazoles elute.
Fig. 4：The electrophoresis detection of the SDS-PAGE of AtUGT73C1.M：Albumen Marker, CS：Smudge cells supernatant, CP：It is broken Broken cell precipitation, FT：Supernatant, 1-4：25mM, 50mM, 100mM, 250mM imidazoles elute.
Fig. 5：The electrophoresis detection of the SDS-PAGE of AtUGT73C5.M：Albumen Marker, CL：Cell pyrolysis liquid, CS：It is broken Cell conditioned medium, CP：Smudge cells precipitate, FT：Supernatant, WB：It is eluted without imidazole buffer and is not associated with albumen, 1-3：15mM, 50mM, 250mM imidazoles elute.
Fig. 6：The electrophoresis detection of the SDS-PAGE of SrUGT76G1.M：Albumen Marker, CL：Cell pyrolysis liquid, CP：It is broken Cell precipitation, CS：Smudge cells supernatant, FT：Supernatant, WB：It is eluted without imidazole buffer and is not associated with albumen, 1-6：15mM, 25mM, 50mM, 100mM, 250mM, 250mM imidazoles elute.
Fig. 7：HPLC analysis glycosyl transferase SrUGT85A8, AtUGT73C1 and AtUGT73C1 is reacted with Steviol's. In figure, the peaks A are located at 22.5min, indicate steviol；The peaks B are located at 12.5min, indicate steviol -13-O- steviosides；The peaks C At 11.3min, instruction generates steviol -19-O- steviosides；The peaks D are located at 5.3min, indicate sweetness agent classification glycosides.
Fig. 8：HPLC analyzes glycosyl transferase SrUGT85A8, AtUGT73C1 and AtUGT73C1 and steviol -19-O- sweet teas The reaction of chrysanthemum glycosides.In figure, the peaks C are located at 11.3min, and instruction generates steviol -19-O- steviosides；The peaks D are located at 5.3min, Indicate sweetness agent classification glycosides.
Fig. 9：HPLC analysis glycosyl transferase SrUGT76G2 are reacted with stevioside.In figure, the peaks E are located at 10.7min Place indicates stevioside；The peaks F are located at 10.2min, indicate content rebaudioside-A.
Figure 10：The mass spectral results that LC/MS analyses AtUGT73C1 is reacted with steviol.A) AtUGT73C1 is reacted with steviol Full wavelength scanner；B) AtUGT73C1 and steviol reaction product steviol -13-O- steviosides, steviol -19-O- steviosides Mass spectral results, [M+Na]+Theoretical value 503.2615, actual value 503.2507；C) AtUGT73C1 and steviol reaction product sweet tea The mass spectral results of leaf raspberry glycosides, [M+Na]+Theoretical value 665.3144, actual value 665.3003.
Figure 11：Mass spectral results a) the AtUGT73C5 that LC/MS analyses AtUGT73C5 is reacted with steviol are reacted with steviol Full wavelength scanner；B) AtUGT73C5 and Steviol reaction product steviol -13-O- steviosides, steviol -19-O- stevia rebaudianums The mass spectral results of glycosides, [M+Na]+Theoretical value 503.2615, actual value 503.2507；C) AtUGT73C5 and steviol reaction product The mass spectral results of sweetness agent classification glycosides, [M+Na]+Theoretical value 665.3144, actual value 665.3003.
Figure 12：The mass spectral results that LC/MS analyses SrUGT85A8 is reacted with steviol.A) SrUGT85A8 is reacted with steviol Full wavelength scanner；B) mass spectral results of SrUGT85A8 and steviol reaction product steviol -19-O- steviosides, [M+Na]+ Theoretical value 503.2615, actual value 503.2507.
Figure 13：The mass spectral results that LC/MS analyses SrUGT76G2 is reacted with steviol glycoside.A) SrUGT76G2 and steviol glycoside Full wavelength scanner, chromatogram and the ion stream of molecular weight of product extraction of reaction；B) SrUGT76G2 and steviol glycoside reaction product The mass spectral results of rebaudioside A, [M+NH4]+Theoretical value 984.4646, actual value 984.4649.
Figure 14：The mass spectral results that LC/MS analyses SrUGT76G1 is reacted with rebaudioside A.A) SrUGT76G1 and Lai Baodi The ion stream of full wavelength scanner, chromatogram and the stevia rebaudianum pentasaccharides product of glycosides A reactions and the sugared molecular weight of product extraction of three stevia rebaudianums six Figure；B) mass spectral results for the stevia rebaudianum pentasaccharides product that SrUGT76G1 is reacted with rebaudioside A, [M+NH4]+Theoretical value 1146.5174, actual value 1146.5150.The mass spectrum knot for the sugared product of three stevia rebaudianums six that SrUGT76G1 is reacted with rebaudioside A Fruit, [M+NH4]+Theoretical value 1380.5703, actual value 1308.5662,1308.5661 and 1308.5648.
Figure 15：The mass spectral results that LC/MS analyses SrUGT76G1 is reacted with Rebaudiodside A I.A) SrUGT76G1 and Lai Baodi Full wavelength scanner, chromatogram and the ion flow graph of the molecular weight of substrate Rebaudiodside A Lay I and product X extraction of glycosides I reactions；b) The mass spectral results of SrUGT76G1 and Rebaudiodside A I reaction products X, [M+NH4]+Theoretical value 1380.5703, actual value 1308.5617。
Figure 16：The structure of steviol and steviol glycoside.
Figure 17：The biosynthetic metabolism approach of stevioside.
Specific implementation mode
The present invention construct respectively glycosyl transferase SrUGT85A8, SrUGT76G1, SrUGT76G2, AtUGT73C1 and The expression vector of AtUGT73C5, and carry out vitro enzyme work test.It was found that the glycosyl transferase AtUGT73C1 in arabidopsis source and AtUGT73C5 can be catalyzed steviol (Steviol) and generate steviol -19-O- steviosides (Steviol-19-O- Glucoside) and steviol -13-O- steviosides (Steviolmonoside), further it is catalyzed steviol -19-O- steviosides Sweetness agent classification glycosides (Rubusoside) is generated, catalytic efficiency is that plant source UGT is relatively high；STEVIA REBAUDIANA source The carboxyl that SrUGT85A8 glycosylates steviol generates steviol -19-O- steviosides, and glycosyl transferase SrUGT76G2 can be urged Changing stevioside and generates content rebaudioside-A, glycosyl transferase SrUGT76G1 can be catalyzed content rebaudioside-A and generate rebaudioside D, into The catalysis of one step generates rebaudioside M2, while SrUGT76G1 can also be catalyzed rebaudioside I and further glycosylate the new change of generation Close object X.Thus the present invention is completed.The present invention these find the further perfect biosynthesis pathway of steviol glycoside；It is available Synthetic biology is used to improve the industrialization of stevioside content by transgenic technology.
Specifically, the present invention, which provides, has such as SEQ ID NO:2, the glycosyl of amino acid sequence shown in 4,6,8 or 10 turns Move enzyme (UGT).The invention also includes in SEQ ID NO:2, with one or several on the basis of amino acid sequence shown in 4,6,8 or 10 A amino acid similar in performance carries out the polypeptide obtained when conservative replaces.This conservative replaces will not usually change Become the function of protein or polypeptide." amino acid similar in performance " includes for example, the amino acid with similar side chain is residual The family of base, these families include the amino acid (such as lysine, arginine, histidine) with basic side chain, have acidity The amino acid (such as aspartic acid, glutamic acid) of side chain, with uncharged polar side chain amino acid (such as glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acid with non-polar sidechain (such as Alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), have β-branched building block Amino acid (such as threonine, valine, isoleucine) and amino acid (such as tyrosine, phenylpropyl alcohol ammonia with aromatic side chain Acid, tryptophan, histidine).
Therefore the polypeptide of the present invention includes SEQ ID NO:2, the mutant of amino acid sequence shown in 4,6,8 or 10, that is, exist SEQ ID NO:2, by replacing, missing or adding one or several amino acid in amino acid sequence shown in 4,6,8 or 10, simultaneously Retain SEQ ID NO:2, the lysine decarboxylase activity that amino acid sequence has shown in 4,6,8 or 10 by SEQ ID NO: 2, polypeptide derived from 4,6,8 or 10.It is described it is several be usually 10 within, within preferably 8, within more preferable 5.
In certain embodiments, polypeptide of the invention also includes UGT74G1 and its remains the mutant of functional activity.
Those skilled in the art can be used conventional technology and judge SEQ ID NO:2,4,6,8,10 and UGT74G1 institutes Which amino acid residue can be substituted or delete in the amino acid sequence shown.For example, by comparison from different genera, active phase With similar or visibly different sequence, it can be determined which amino acid residue is can be substituted or delete in these sequences. Whether the such sequence of method (including the method disclosed in the present) verification that this field routine can be used has institute of the present invention The enzyme activity stated.
In addition, it is well known to those skilled in the art, in gene cloning operation, it is often necessary to suitable restriction enzyme site is designed, This certainly will introduce one or more incoherent residues in expressed albumen end, and this has no effect on the work of destination protein Property.For another example, the recombination egg being secreted into automatically for construction of fusion protein, the expression of promotion recombinant protein, acquisition outside host cell In vain or conducive to recombinant protein purifying, it is often necessary to by some amino acid or amino acid sequence be added to recombinant protein N end In other appropriate areas in end, C-terminal or the albumen, these amino acid sequences include but not limited to the joint peptide being suitble to, letter Number peptide, leader peptide, end extension, glutathione S-transferase (GST), maltose E binding protein, albumin A or Xa factor are solidifying The proteolytic enzyme site of hemase or enterokinase, and can be to the protein tag that albumen is purified, such as FLAG, HA, HA1, c- Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1 etc..It should be understood that these ammonia The presence of base acid sequence does not interfere with the activity of gained polypeptide.Therefore, the present invention is also included within polypeptide of the present invention (including SEQ ID NO:2, including 4,6,8 and 10) C-terminal and/or N-terminal add the polypeptide obtained by one or several amino acid, these are more Peptide still has glycosyl transferase activity as described herein.
Therefore, the present invention also includes and SEQ ID NO:2, amino acid sequence shown in 4,6,8,10 or UGT74G1 has 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 The amino acid sequence of 99% sequence identity.In a more preferred embodiment, such amino acid sequence similarly comes from STEVIA REBAUDIANA Or arabidopsis, preferably have and SEQ ID NO:2, the same or similar glycosyl transferase enzyme activity of 4,6,8,10 or UGT74G1, such as There is same or similar kinetic parameter (such as Km, Kcat and Kcat/Km value) under identical reaction system and reaction condition.
The sequence identity that the two sequences of conventional means calculating ratio pair can be used, for example, being provided using NCBI BLASTP is compared using default parameters.
The polypeptide of the present invention can be native purified product or chemically synthesized product, or using recombinant technique from It is generated in protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).
The application includes polynucleotide sequence, and the polynucleotide sequence is selected from the polynucleotides sequence for encoding polypeptide of the present invention Row and complementary series.SEQ ID NO:1,3,5, the 7 and 9 exemplary coding sequence for showing polypeptide of the present invention.The present invention's is more Nucleotide sequence also includes the polynucleotide sequence with the coded sequence and complementary series very high homology, or under strict conditions The polynucleotide sequence of hybridization, or with SEQ ID NO:1, the family gene molecule of 3,5,7 or 9 very high homologies.The present invention also wraps The long 10-40 base of polynucleotide sequence of the present invention, the segment of preferably 15-30 base are included, these segments can be used as visiting Needle or primer.Herein, " segment " refers to continuous a part of sequence of full length sequence herein.
The sequence of coding polypeptide of the present invention includes：The coded sequence of encoding mature polypeptide；The coded sequence of mature polypeptide With various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding sequence of mature polypeptide.
The polynucleotide sequence or its segment of the present invention can usually use PCR amplification method, recombination method or artificial synthesized side Method obtains.
The present invention also relates to nucleic acid constructs, which contains the polynucleotide sequence and more with this of the present invention Nucleotide sequence is operatively connected and guide the polynucleotide sequence to be expressed under suitable conditions in host cell one Or multiple regulating and controlling sequences.The polynucleotide sequence of coding polypeptide of the present invention can be operated in various ways, to ensure the polypeptide Expression.Before it is inserted into carrier the operation of the polynucleotide sequence may according to the expression vector but cater to the need or required 's.The technology for changing polynucleotide sequence using recombinant DNA method is known in the art.
Regulating and controlling sequence can be suitable promoter sequence, be by for expressing the polynucleotides for encoding polypeptide of the present invention The nucleotide sequence of host cell identification.Promoter sequence includes the transcription regulating nucleotide sequence for being connected to polypeptide expression.Promoter can be with Be any nucleotide sequence of transcriptional activity is shown in selected host cell, including mutation, truncated and heterozygosis opens Mover, and can be obtained from the gene for encoding the extracellular or intracellular polypeptide homologous or heterologous with the host cell.Suitable for this The promoter sequence example of invention includes 35S promoter and cspA promoters etc..
Regulating and controlling sequence can also be suitable transcription terminator sequences, to be identified by host cell to terminate the sequence of transcription Row.Terminator sequence and 3 ' ends of the nucleotide sequence for encoding the polypeptide are operatively connected.Have in the host cell of selection Any terminator of function can be used in the present invention.
Regulating and controlling sequence can also be suitable targeting sequencing, the non-translational region of the mRNA important to host cell translation.Label It is operatively connected to sequence and 5 ' ends of the nucleotide sequence for encoding the polypeptide.Functional in the host cell of selection What terminator can be used in the present invention.
Regulating and controlling sequence can also be the amino acid sequence of the amino-terminal end connection of coding and polypeptide and instruct the polypeptide Into the signal peptide coding region of cell secretory pathway.5 ' ends of nucleotide sequence coded sequence can include inherently natural signal Peptide-coding region.Alternatively, 5 ' ends of coded sequence may include the signal peptide coding region with the code area external source.Optionally, external Signal peptide coding region can simply replace natural signal peptide coding region to enhance the secretion of polypeptide.
The present invention also relates to cloning vectors or expression vector including polynucleotides of the present invention.These carriers can contain above Each regulating and controlling sequence.
Expression vector can be easily subjected to recombinant DNA method and can lead to interested nucleotides sequence list Any carrier (such as plasmid or virus) reached.It is thin with the host that is wherein imported into the carrier that the selection of carrier is generally dependent on carrier The compatibility of born of the same parents.The carrier can be circular plasmids that are linear or being closed.
Carrier can be the carrier of autonomous replication, that is, be used as extrachromosomal entity to exist, and replicate independent of chromosome The carrier of duplication, such as plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier may include for ensureing certainly Any mode that I replicates.Alternatively, carrier can be when being imported into host cell, be integrated into genome and with it The carrier that chromosome through being be integrated into replicates together.In addition, it includes that will be imported into host cell gene group together that can be used Total DNA single carrier or plasmid or two or more carriers or plasmid or transposons.
The carrier of the present invention preferably comprises one or more, and to allow to easily choose conversion, transfection, transduction isocellular optional Select label.Selectable label is gene, and product provides the resistance to antibiotic or virus, the resistance to heavy metal, original and supports Type is to auxotroph etc..
The carrier of the present invention, which preferably comprises, allows the vector integration to enter host cell gene group or the carrier in cell Independently of autonomous element replicated of genome.
The polynucleotides of the present invention of more than one copy can be inserted into host cell to increase the yield of the gene outcome. Increasing for polynucleotide copies number can be by being integrated into host cell gene group by the sequence of at least one additional copies or leading to It crosses and is obtained including amplifiable selectable marker gene and the polynucleotides, wherein the selectable marker gene comprising amplification copy is simultaneously And the cell thus comprising additional copies polynucleotides can be screened by cultivating the cell when there are selective agent appropriate.
The carrier of the present invention preferably comprises one section of artificial synthesized sequence, contains multiple limitation endonuclease recognized sites, energy A variety of pluggable positions or interleaved plan are provided for exogenous DNA.The expression vector of the present invention is more highly preferred to include continuous 6 The small peptide of histidine sequences is conducive to the extraction and purifying of protein.
Cloning vector containing polynucleotide sequence of the present invention can be used for replicating enough target plasmids.Therefore, this hair Bright cloning vector carries stronger self-replacation element, such as replication origin.In general, the cloning vector of the present invention does not have There is Expression element.
The present invention also relates to the recombinant host cells containing the polynucleotides of the present invention for being used for recombinant production polypeptide.Including The carrier of polynucleotides of the present invention is imported into host cell so that the composition portion as chromosome of the carrier as explained earlier Divide or is maintained as extrachromosomal self-replacation carrier.The selection of host cell is heavily dependent on coding polypeptide Gene and its source.
Host cell can be plant cell or unicellular microorganism or non-unicellular micro-organism.Host cell can be former Nucleus, including saccharomyces, pseudomonas (Pseudomonas), bacillus (Bacillus), Enterobacter (Enterobacter), staphylococcus (Staphylococcus), streptomyces (Streptomyces) and Escherichia Belong to the bacterium of (Escherichia).In certain embodiments, host cell is plant cell, such as STEVIA REBAUDIANA source is thin The cell in born of the same parents or arabidopsis source.In certain embodiments, host cell is Escherichia coli or yeast.
In certain aspects, host cell of the invention is Agrobacterium, and the Agrobacterium contains expression polypeptide of the present invention Expression vector.Agrobacterium can be Agrobacterium well known in the art, such as Agrobacterium tumefaciems and agrobacterium rhizogenes.
Conventional transfection method can be used the nucleic acid constructs containing polynucleotide sequence of the present invention is transferred in host cell. Transfection is generally divided into transient transfection and stable transfection.In the former exogenous DNA/RNA unconformity to host chromosome, therefore a place Multiple copy numbers may be present in chief cell, generate high-caliber expression, but usually only last for several days.In stable transfection, exogenous DNA Both it can be integrated into host chromosome, it is also possible to exist as a kind of episome.The technological means of transfection includes chemical transfection And physical transfection, the former such as DEAE- glucans method, calcium phosphate method and artificial liposome method, the latter such as microinjection, electroporation and Particle gun etc..
It should be understood that the host cell of the present invention can only express SEQ ID NO:2 and/or its remain glycosyl transferase Active mutant, SEQ ID NO:4 and/or its remain the mutant of glycosyl transferase activity, SEQ ID NO:6 and/or It remains the mutant of glycosyl transferase activity or SEQ ID NO:8 and/or its remain the mutation of glycosyl transferase activity Body or SEQ ID NO:10 and/or its remain the mutant of glycosyl transferase activity, or SEQ ID NO can be expressed:2 and/or It remains the mutant of glycosyl transferase activity, SEQ ID NO:4 and/or its remain the mutation of glycosyl transferase activity Body, SEQ ID NO:6 and/or its remain the mutant of glycosyl transferase activity, SEQ ID NO:8 and/or its remain sugar The active mutant of based transferase and SEQ ID NO:10 and/or its remain it is arbitrary in the mutant of glycosyl transferase activity Two kinds, arbitrary three kinds or four kinds of whole, such as express SEQ ID NO simultaneously:6 and/or its remain glycosyl transferase activity Mutant and SEQ ID NO:8 and/or its remain the mutant of glycosyl transferase activity；Or SEQ ID NO are expressed simultaneously:4 With/it remains the mutant of glycosyl transferase activity, SEQ ID NO:6 and/it remains the mutation of glycosyl transferase activity Body and SEQ ID NO:8 and/it remains the mutant of glycosyl transferase activity, or also expression SEQ ID NO:2 and/or its Remain the mutant of glycosyl transferase activity.In certain embodiments, stevioside biology well known in the art can also be closed It is transferred at the expression vector of other enzymes in metabolic pathway in the host cell of the present invention, this fermentoid includes but not limited to UGT74G1 (the numbers of logging in：AY345982).
After the coded sequence for obtaining polypeptide, following method can be used and produce polypeptide of the present invention, this method includes：(a) exist Contribute under conditions of production polypeptide to cultivate host cell；And (b) recycle the polypeptide.
In the production method of the present invention, cell can be used a method known in the art in the culture medium suitable for producing polypeptide Middle culture.For example, cell can be by the shaking flask culture that is carried out in laboratory or industrial fermentation tank and small-scale or large-scale Fermentation (including it is continuous, in batches, batch feeding or solid state fermentation), suitable culture medium and allow the polypeptide expression and/ Or it is cultivated under conditions of separation.Culture, which is happened at, to be used a method known in the art including carbon source and nitrogen source and inorganic salts In suitable culture medium.Suitable culture medium is available from commercial provider or can be prepared according to disclosed composition.If should Polypeptide secretion enters culture medium, which can directly recycle from culture medium.If the polypeptide is not secreted into culture medium, it can be from Cell lysate recycles.
Polypeptide described in the invention can be recycled using methods known in the art.For example, polypeptide can pass through conventional side Method, including but not limited to centrifugation, filtering, ultrafiltration, extraction, chromatography, spray drying, freeze-drying, evaporation or precipitation etc. are from culture Base recycles.
The present invention polypeptide can be purified by a variety of methods known in the art, including but not limited to chromatography (such as from Sub- exchange, compatibility, hydrophobicity, chromatofocusing, molecular exclusion), electrophoresis (such as isoelectric focusing), differential solubility (such as salt Analysis precipitation), SDS-PAGE or extraction to be to obtain substantially pure polypeptide.
The polypeptide of the present invention can be used for the preparation of stevioside, such as steviol -13-O- steviosides, steviol -19- The biosynthesis of O- steviosides, sweetness agent classification glycosides, stevioside and/or content rebaudioside-A.For example, using the table of the present invention Up to the engineered strain of vector construction biosynthesis pathway containing stevioside, stevioside biosynthesis way is produced via the engineered strain One or more multi-products in diameter.Figure 17 shows the biosynthetic metabolism approach of stevioside.Thus, for example, SEQ ID NO:4,6,8 or its remain in the mutant of glycosyl transferase activity one or more can be used for steviol -19-O- stevia rebaudianums The synthesis of glycosides or sweetness agent classification glycosides；SEQ ID NO:6,8 or its remain one kind in the mutant of glycosyl transferase activity Or a variety of synthesis that can be used for steviol -13-O- steviosides.And SEQ ID NO:10 and/or its remain glycosyl transferase work Property mutant can be used for glycosylate content rebaudioside-A to synthesize rebaudioside D, rebaudioside M2, rebaudioside M and Lai Baodi Glucoside I.And SEQ ID NO:2 and/or its remain the mutant of glycosyl transferase activity then can be used for glycosylate stevioside with Content rebaudioside-A is synthesized, and can be used for glycosylating rebaudioside I to synthesize compound X.
Therefore, engineered strain of the invention can express SEQ ID NO:2,4,6,8,10 or its remain glycosyl transferase work It is one or more in the mutant of property, or containing SEQ ID NO can be expressed:2,4,6,8,10 or its remain glycosyl transferase It is one or more in the expression vector of active mutant.In certain embodiments, engineered strain of the invention at least table Up to SEQ ID NO:2 or its remain the mutant of glycosyl transferase activity, while also expression SEQ ID NO:4,6,8,10 or Its remain it is one or more in the mutant of glycosyl transferase activity, such as SEQ ID NO:4,6 and 8 or their reservation The mutant of glycosyl transferase activity, or also further expression SEQ ID NO:10 or its remain glycosyl transferase activity Mutant.Alternatively, the engineered strain of the present invention, which at least contains, can express SEQ ID NO:2 or its remain glycosyl transferase activity Mutant expression vector, while also containing expression SEQ ID NO:4,6,8,10 or its remain glycosyl transferase activity It is one or more in the expression vector of mutant, such as express SEQ ID NO:4,6 and 8 or they remain glycosyl transferase The expression vector of active mutant, or also further contain expression SEQ ID NO:10 or its remain glycosyl transferase activity Mutant expression vector.
Preferably, other enzymes in stevioside biosynthesis pathway are also expressed in engineered strain of the invention, such as UGT74G1.The example that can be used as engineered strain of the present invention includes but not limited to Escherichia coli and yeast etc..
In certain embodiments, the present invention relates to it is a kind of structure genetically modified plants method, the method includes：
(1) Agrobacterium for carrying expression vector is provided, the expression vector expresses polypeptide of the present invention；
(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), makes the code sequence of polypeptide of the present invention Row are transferred to plant cell, and are integrated on the chromosome of plant cell；
(3) plant cell or tissue for being transferred to the coded sequence are selected；With
(4) by step (3) plant cell or regeneration at plant.
It should be understood that the expression vector being transferred in the plant cell can express a kind of polypeptide of the present invention, it also can table Up to two or more polypeptide.Therefore, one or more expression vectors can be transferred in the plant cell, so that institute's structure The genetically modified plants built can synthesize one or more products or whole products in stevioside biosynthesis pathway.
Preferably, this kind of genetically modified plants are the plant of natural synthesis stevioside, such as STEVIA REBAUDIANA.Through the invention The yield of stevioside in the plant can be improved in transgenic method.Alternatively, this kind of genetically modified plants can be suitable for industrial metaplasia The plant of stevioside is produced, after the carrier of transgenic method transfer expression polypeptide of the present invention through the invention, this hair can be expressed Bright polypeptide and one or more products in biosynthesis stevioside biosynthesis pathway or whole products.
In certain embodiments, the present invention provides plant tissue, and the cell contained by the plant tissue contains the present invention Expression vector, express the present invention one or more polypeptides.Plant tissue can be various plant tissues well known in the art, Such as callus, seed or a part for plant.In certain embodiments, a part for the plant can not regenerate plant Strain.For example, the present invention plant tissue be STEVIA REBAUDIANA leaf, the leaf be rich in stevioside biosynthesis pathway in one kind or Multi-products or whole products.The sweet tea in the blade is extracted using water extraction method and organic solvent extraction well known in the art One or more products in chrysanthemum glycosides biosynthesis pathway or whole products.
In certain embodiments, the present invention also provides the compound X with formula：
Compound X acid and alkali-resistances, are dissolved in water, can be used as sweetener.The SEQ ID NO of the present invention can be used:10 or its reservation The mutant glycosylation rebaudioside I of glycosyl transferase activity and obtain compound X.
The present invention will be hereafter illustrated in a manner of specific embodiment.The experiment side of actual conditions is not specified in the following example Method, such as Sambrook usually according to normal condition,《Molecular cloning：Lab guide》(New York, United States：Cold spring harbor laboratory Publishing house, 1989) condition described in, or carry out according to the normal condition proposed by manufacturer.For the usage and dosage of reagent, remove It is non-to be otherwise noted, otherwise used according to conventional usage and dosage.
Embodiment 1：The extraction and detection of total serum IgE
Super-clean bench and ultraviolet lamp are opened, burns mortar, key, scissors with ethyl alcohol.Blade 100mg is cut, by tissue in liquid nitrogen Grind into powder in packing to Ep pipes, adds 1ml Trizol-x-100 after liquid nitrogen volatilization, firmly shakes or blown with pipettor immediately 5-8 times (without until block) is inhaled, 5min is stored at room temperature；With isometric chloroform 2 times, 7500g centrifuges 15min；Supernatant The isopropanol being pre-chilled in advance in equal volume is added in liquid, and 30min is placed at room temperature for after mixing, and 4 DEG C of 10000g centrifuge 10min；Precipitation is added 75% ethyl alcohol of 1ml is cleaned, and 4 DEG C of 10000g centrifuge 10min；It is dissolved in 25 μ l DEPC processing after precipitation drying at room temperature 10min Water in, with 1.0% agarose gel electrophoresis detect RNA integrality, with Eppendorf nucleic acid quantification instrument measure A260, A280 ratios and concentration.It is spare to be placed in -80 DEG C of refrigerators.
Embodiment 2：The structure of recombinant bacterial strain
(1) reverse transcription synthesizes cDNA
The PrimeScript reverse transcription reagent box provided using TakaRa companies, is prepared total using embodiment 1 RNA synthesizes STEVIA REBAUDIANA first complementary strand of mRNA.
(2) clone of glycosyltransferase gene
Stevia based transferase SrUGT85A8 (nucleotide sequence such as SEQ ID NO are searched for from NCBI:Shown in 3, ammonia Base acid sequence such as SEQ ID NO:Shown in 4), arabidopsis AtUGT73C1 (nucleotide sequence such as SEQ ID NO:Shown in 5, amino acid Sequence such as SEQ ID NO:Shown in 6), arabidopsis AtUGT73C5 (nucleotide sequence such as SEQ ID NO:Shown in 7, amino acid sequence Such as SEQ ID NO:Shown in 8) and stevia based transferase SrUGT76G1 (nucleotide sequence such as SEQ ID NO:Shown in 9, Amino acid sequence such as SEQ ID NO:Shown in 10) CDS sequences, while STEVIA REBAUDIANA transcript profile data are analyzed, find one Item has the new glycosyl transferase of potential function, is named as SrUGT76G2 (nucleotide sequence such as SEQ ID NO:Shown in 1, amino Acid sequence such as SEQ ID NO:Shown in 2).
It is analyzed by comparing, using vector NTI Software for Design 4 to amplimer as shown in Table 1.With STEVIA REBAUDIANA,
The cDNA of arabidopsis is template, according to following system (5 × phusion buffer solutions 4.0 μ l, dNTP (2.5mM) 1.6 μ L, each 0.6 0.5 μ L, Phusion archaeal dna polymerase of μ l, cDNA of 2 μ L, DMSO, 0.2 μ L of primer (2 μM), adds ddH2O is to whole body 20.0 μ L of product) amplification glycosyltransferase gene, response procedures：98 DEG C of pre-degenerations 30s, 98 DEG C of denaturation 10s, 55 DEG C of 30s that anneal, 72 DEG C extend 2min, 30 cycle, 72 DEG C eventually extend 5min, 16 DEG C preservation.Amplification is detected with 1% agarose gel electrophoresis, The results are shown in Figure 1.
Using the Agarose Gel Fragment Recovery Kit Ver.2.0 purified pcr products of Axygen companies,
Then pMD19-T carriers are cloned into, the plasmid of screening positive clone extraction send Sani company to be sequenced, and obtains glycosyl and turns Move enzyme related gene sequence.
Table 1：Primer (SEQ ID NO needed for glycosyl transferase amplification:11-20)
(3) structure of glycosyl transferase prokaryotic expression carrier
Using the glycosyltransferase gene in carrier T as template, PCR amplification is carried out using the primer of table 1, amplified production is through electricity Swimming detection, with restriction enzyme in 37 DEG C of digestion 2h, enzyme is purified using plastic recovery kit (Axygen companies) with after purification Cut product.SrUGT85A8 is connected to pET-28a (+) through restriction enzyme BamHI and NcoI after 37 DEG C of digestion 2h, SrUGT76G2 is connected to pET-28a (+), AtUGT73C1 warps through restriction enzyme BamHI and NotI after 37 DEG C of digestion 2h Restriction enzyme BamHI and NotI is connected to pET-28a (+) after 37 DEG C of digestion 2h, and AtUGT73C5 is through restriction enzyme BamHI and NotI is connected to pET-28a (+) after 37 DEG C of digestion 2h, and SrUGT76G1 is through restriction enzyme BamHI and NotI PET-28a (+) is connected to after 37 DEG C of digestion 2h.
(4) structure of recombinant bacterial strain
Under the action of DNA ligase, the PCR fragment after respectively recycling double digestion is reacted with carrier segments in 16 DEG C 12h, connection product utilize CaCl2Method converts e. coli bl21 (DE3), is screened on kalamycin resistance tablet, and Full bacterium PCR detections are carried out to clone, positive clone molecule is accessed in LB liquid medium and is cultivated, recombinant plasmid is extracted, is carried out BamHI and NotI double digestions are verified, and there are two segments after agarose gel electrophoresis identification complete degestion, show expression vector structure Build up work(, be respectively designated as pET-28a (+)/SrUGT85A8, pET-28a (+)/SrUGT76G2, pET-28a (+)/ AtUGT73C1, pET-28a (+)/AtUGT73C5 and pET-28a (+)/SrUG76G1.
Embodiment 3：The expression of glycosyltransferase gene
The positive clone molecule that the screening of embodiment 2 obtains is inoculated into the LB liquid containing that resistance (100 μ g/mL of final concentration) of card In body culture medium, cell density is detected using spectrophotometer after 37 DEG C of 150r/min cultures 3-4h, is added when OD600 is about 0.6 Enter IPTG (final concentration 0.2mM) induced expression, take 1mL bacterium solutions in 1.5mL centrifuge tubes every 2h, thalline were collected by centrifugation, respectively Isometric 2X sample-loading buffers are added, are analyzed using 10% SDS-PAGE electrophoresis detections after the of short duration centrifugations of boiling water bath 5min. Compared with empty carrier recombinant bacterial strain [pET-28a (+)], the glycosyltransferases expression carrier of recombination has more a protein band, and And with the increase of induction time, the expression quantity of glycosyltransferase gene gradually increases.
Embodiment 4：The purifying of glycosyltransferase gene expression vector and activity identification
(1) purifying of glycosyltransferase gene expression vector
Mass propgation recombinant bacterial strain is collected thalline, is used Tris buffer solutions (100mM, pH8.0) through IPTG induced expressions Thalline is resuspended, lysozyme dissolving, ice bath squeezer smudge cells, 18000rpm centrifugations 30min collects supernatant, according to fusion protein On His-Tag using Ni-NTA purifying recombination glycosyl transferase, be utilized respectively various concentration imidazole solution elution purpose egg In vain, refined solution, 10% SDS-PAGE electrophoresis detection purification effects are collected.As a result as shown in Figure 2-5.
(2) activity identification of glycosyltransferase gene expression vector
Enzyme reaction system is prepared in the Tris buffer solutions (100mM, pH8.0), makes each Reagents Final Concentration be respectively：BSA (0.01mg/ml)、UDPG(2mM)、MgCl2(5mM), substrate (1mM) and step (1) purify obtained various glycosyl transferases (30μM).Reaction system is placed in 37 DEG C of water-bath 2h.
HPLC is detected：Reaction solution is extracted with the 300 μ l of n-butanol of saturation 2 times, and spiral concussion, 12000rpm centrifuges 10s, just Butanol phase centrifugal concentrating, after dissolved with 50%MeOH.Using Phenomenex C18,4.6 × 250mm, 4 μm of (p/n 00G- 4375-E0)；Column temperature：30℃；Mobile phase A：0.1% formic acid, organic phase B：Acetonitrile；Flow velocity：1.0mL/min；Sample size：20μL； Ultraviolet detection (210nm).
As a result as shown in Fig. 7-15.
Fig. 7's the results show that the carboxyl of STEVIA REBAUDIANA SrUGT85A8 glycosylation steviols (Steviol) generates steviol- The structure of 19-O- steviosides (Steviol-19-O-Glucoside), steviol -19-O- steviosides is verified through two-dimentional nuclear-magnetism；It is quasi- Southern mustard AtUGT73C1 and AtUGT73C5 can glycosylate steviol (Steviol) and generate steviol -19-O- steviosides, sweet tea Alantol -13-O- steviosides (Steviolmonoside) and sweetness agent classification glycosides (Rubusoside), the structure of three products are equal It is verified by nuclear-magnetism.
Fig. 8's the results show that STEVIA REBAUDIANA SrUGT85A8 cannot glycosylate steviol -19-O- steviosides；Arabidopsis AtUGT73C1 and AtUGT73C5 can glycosylate steviol -19-O- steviosides and generate sweetness agent classification glycosides, product structure warp Cross nuclear-magnetism verification.
The HPLC of Fig. 9 generates content rebaudioside-A, product knot the results show that STEVIA REBAUDIANA SrUGT76G2 can glycosylate stevioside Structure is verified through nuclear-magnetism.
Figure 10-12 respectively illustrate LC/MS analysis AtUGT73C1, AtUGT73C5 and SrUGT85A8 and steviol it is anti- The mass spectral results answered.
Figure 13 shows the mass spectral results that LC/MS analyses SrUGT76G2 is reacted with steviol glycoside.
Figure 14 shows the mass spectral results that LC/MS analyses SrUGT76G1 is reacted with rebaudioside A.
Figure 15 shows the mass spectral results that LC/MS analyses SrUGT76G1 is reacted with Rebaudiodside A I.
Sequence table
<110>Shanghai Inst. of Life Science, CAS
<120>Glycosyl transferase and its application
<130> 168067
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 1563
<212> DNA
<213>STEVIA REBAUDIANA（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 atggatccgc 1440
gacccatttg ctgtccacca gtcatgctag ccatatggct gccgcgcggc accaggccgc 1500
tgctgtgatg atgatgatga tggctgctgc ccatggtata tctccttctt aaagttaaac 1560
aaa 1563
<210> 2
<211> 470
<212> PRT
<213>STEVIA REBAUDIANA（Stevia rebaudiana (Bertoni) Hemsl）
<400> 2
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Glu
1 5 10 15
Asn Gln Pro Gln Thr Thr Val Arg Arg His Arg Arg Ile Ile Leu Phe
20 25 30
Pro Met Pro Phe Gln Gly His Ile Asn Pro Met Ile Gln Leu Ala Asn
35 40 45
Leu Leu Tyr Ser Asn Gly Phe Ser Ile Val Ile Leu His Thr Asn Phe
50 55 60
Asn Ala Pro Lys Phe Ser Asn Tyr Pro Asn Phe Thr Phe Ile Ser Val
65 70 75 80
Leu Asp Asn Ala Glu Asn Glu Pro Phe Ser Thr Ser Ser Ser Phe Glu
85 90 95
Phe Ala Gln Asn Tyr Thr Phe Lys Gln Asp Gly Ala Asp Glu Leu Arg
100 105 110
His Lys Leu Glu Leu Leu Leu Ala Ser Gly Asn Asp Glu Pro Val Ser
115 120 125
Cys Leu Ile Thr Asp Ala Ile Trp His Phe Thr Gln Ser Val Ala Asp
130 135 140
Ser Leu Gln Ile Pro Arg Ile Val Leu Arg Thr Thr Ser Val Tyr Cys
145 150 155 160
Ser Ile Val Tyr Ala Ser Ile Pro Leu Phe Asp Asp Arg Gly Tyr Phe
165 170 175
Ala Leu Asp Asp Ser His Leu Glu Glu Gln Val Met Glu Phe Pro Leu
180 185 190
Leu Lys Val Lys Asp Ile Lys Lys Ile Gly Ile Lys Ser Met Asn Asp
195 200 205
Pro Tyr Ala Lys Met Ile Arg Glu Met Val Lys Gln Ile Lys Ala Ser
210 215 220
Ser Gly Ile Ile Trp Asn Ser Phe Lys Glu Leu Glu Glu Thr Glu Leu
225 230 235 240
Asp Ser Ile Pro His Asp Phe Pro Ile Pro Arg Phe Ile Ile Pro Phe
245 250 255
His Lys Tyr Phe Asn Ala Ser Ser Ser Ser Leu Leu Glu Gln Asp Arg
260 265 270
Thr Ile Phe Pro Trp Leu Asp Gln Gln Ala Pro Lys Ser Val Leu Tyr
275 280 285
Val Ser Phe Gly Ser Leu Cys Gln Val Asp Glu Lys Glu Phe Leu Glu
290 295 300
Ile Ala His Gly Leu Val Tyr Ser Gln Gln Pro Phe Leu Trp Val Val
305 310 315 320
Arg Leu Gly Phe Val Lys Asp Ser Thr Trp Leu Glu Ser Leu Pro Leu
325 330 335
Pro Asp Gly Phe Pro Gly Glu Arg Gly Arg Val Val Lys Trp Ala Pro
340 345 350
Gln Gln Glu Val Leu Ala His Glu Ala Thr Gly Ala Phe Trp Thr His
355 360 365
Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu Gly Val Ala Met
370 375 380
Ile Cys Ser Pro Phe Trp Gly Asp Gln Pro Ile Asp Ala Arg Tyr Met
385 390 395 400
Ser Asp Val Ser Lys Val Gly Val Tyr Leu Glu Asn Gly Phe Gln Arg
405 410 415
Asp Glu Ile Ser Ser Ala Ile Arg Arg Val Met Val Asp Glu Asp Gly
420 425 430
Lys Asp Ile Arg Glu Arg Val Lys Val Leu Lys Gln Lys Val Asp Asp
435 440 445
Ser Leu Met Ile Ser Gly Ser Ser Asn Glu Ser Leu Glu Ser Leu Ile
450 455 460
His Tyr Ile Ser Ser Phe
465 470
<210> 3
<211> 1576
<212> DNA
<213>STEVIA REBAUDIANA（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（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（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（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（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（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（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>Arabidopsis（Arabidopsis thaliana）
<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 (10)

1. a kind of polypeptide, which is characterized in that the polypeptide is selected from：
(1)SEQ ID NO:Polypeptide shown in 2；With
(2) by SEQ ID NO:Amino acid sequence shown in 2 and promotion SEQ ID NO:The expression of amino acid sequence shown in 2 divides Secrete and/or purify amino acid or amino acid sequence composition polypeptide.
2. a kind of polynucleotide sequence, which is characterized in that the polynucleotide sequence is selected from：
(1) polynucleotide sequence of polypeptide described in claim 1 is encoded；
(2) with the polynucleotide sequence of (1) described polynucleotide sequence complementation；With
(3) segment of the long 10-40 base of (1) or (2) described polynucleotide sequence.
3. polynucleotide sequence as claimed in claim 2, which is characterized in that the polynucleotide sequence is selected from：
(a)SEQ ID NO:Polynucleotide sequence shown in 1；
(b)SEQ ID NO:1 complementary series；With
(c)SEQ ID NO:1 or its complementary series long 10-40 base segment.
4. a kind of nucleic acid constructs, which is characterized in that the nucleic acid constructs contains polynucleotides according to claim 2 or 3 Sequence；
Preferably, the nucleic acid constructs is cloning vector or expression vector.
5. a kind of genetically engineered host cell, which is characterized in that the host cell：
(1) polypeptide described in claim 1 is expressed；And/or
(2) containing the nucleic acid constructs described in claim 4；
Preferably, the host cell is plant cell or Agrobacterium.
6. host cell as claimed in claim 5, which is characterized in that the host cell is also：
(a) expression SEQ ID NO:4,6,8,10 or its remain it is one or more in the mutant of glycosyl transferase activity；It is excellent Selection of land, the host cell at least express SEQ ID NO:4,6 and 8 or its remain the mutant of glycosyl transferase activity, and/ Or
(b) contain expression SEQ ID NO:4,6,8,10 or its remain in the expression vector of the mutant of glycosyl transferase activity It is one or more；Preferably, the host cell at least contains expression SEQ ID NO:4,6 and 8 or its remain glycosyl turn Move the expression vector of the mutant of enzymatic activity.
7. polypeptide described in claim 1 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:4 or its reservation The mutant of glycosyl transferase activity, SEQ ID NO:6 or its remain the mutant SEQ ID of glycosyl transferase activity NO:8 or its remain the mutant of glycosyl transferase activity and SEQ ID NO:10 or its remain glycosyl transferase activity Mutant in one or more, coded sequence, expression vector, expression polypeptide described in claim 1 or its remain Mutant, the SEQ ID NO of glycosyl transferase activity:4 or its remain the mutant of glycosyl transferase activity, SEQ ID NO: 6 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:8 or its remain the mutant of glycosyl transferase activity And SEQ ID NO:10 or its remain genetically engineered place one or more in the mutant of glycosyl transferase activity Application of the chief cell in preparing stevioside.
8. application selected from the following：
(1)SEQ ID NO:4 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:6 or its remain glycosyl The mutant and SEQ ID NO of transferase active:8 or its remain the mutant of glycosyl transferase activity, coded sequence, Expression vector, or expression SEQ ID NO:4 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:6 or its guarantor The mutant and SEQ ID NO of glycosyl transferase activity are stayed:8 or its remain one in the mutant of glycosyl transferase activity The application of kind or a variety of genetically engineered host cells in synthesis steviol -19-O- steviosides or sweetness agent classification glycosides；
(2)SEQ ID NO:6 or its remain the mutant of glycosyl transferase activity and/or SEQ ID NO:8 or its remain The mutant of glycosyl transferase activity, coded sequence, expression vector or SEQ ID NO:6 or its remain glycosyl transferase Active mutant and/or SEQ ID NO:8 or its remain the genetically engineered place of the mutant of glycosyl transferase activity Application of the chief cell in synthesizing steviol -13-O- steviosides；And
(3) polypeptide described in claim 1 or its remain the mutant of glycosyl transferase activity, coded sequence, expression carry Body, or expression polypeptide described in claim 1 or its remain the genetically engineered place of the mutant of glycosyl transferase activity Application of the chief cell in synthesizing content rebaudioside-A.
9. a kind of method of structure genetically modified plants, the method includes：
(1) Agrobacterium for carrying expression vector is provided, the expression vector expresses polypeptide described in claim 1 or it is remained Mutant, the SEQ ID NO of glycosyl transferase activity:4 or its remain the mutant of glycosyl transferase activity, SEQ ID NO: 6 or its remain the mutant of glycosyl transferase activity, SEQ ID NO:8 or its remain the mutant of glycosyl transferase activity And SEQ ID NO:10 or its remain it is one or more in the mutant of glycosyl transferase activity；
(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), so that the coded sequence is transferred to plant thin Born of the same parents, and be integrated on the chromosome of plant cell；
(3) plant cell or tissue for being transferred to the coded sequence are selected；With
(4) by step (3) plant cell or regeneration at plant.
10. using a part for the regeneration plant that claim 9 the method obtains, such as leaf.
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