Glycosyl transferase gene as well as coding product and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and relates to a stevioside glycosyl transferase gene related to stevioside biosynthesis as well as a coding product and application of the stevioside glycosyl transferase gene. The gene is cloned from stevia rebaudiana for the first time, the nucleotide sequence of the gene is as shown in SEQ ID NO.2, and the coded amino acid sequence of the gene is as shown in SEQ ID NO.1. The stevioside glycosyl transferase disclosed by the invention has the capability of carrying out specific xylosylation on a C19 site, the capability of carrying out glycosylation on a C13 site is relatively weak, the activity on a substrate with monosaccharide glycosylation on the C13 site is relatively high, and the activity on glucoside containing a plurality of glycosyls on the C13 site is relatively weak. Overexpression of the glycosyl transferase in stevia rebaudiana can provide a new direction for stevia rebaudiana variety improvement.

Description

Technical Field

[0001] The present invention relates to a gene of UDP - glycosyltransferases (UGT) related to steviol glycoside biosynthesis, its encoded product and applications, belonging to the field of plant genetic engineering. Background Art

[0002] Steviol glycosides (SGs) are a class of diterpene saponin natural products extracted from Stevia rebaudiana, which have the characteristics of high sweetness and low calories. They are known as the "third sugar source in the world" after sucrose and beet sugar, and also have a wide range of biological activities, including anti - inflammatory, antibacterial and hypoglycemic effects. Due to the relatively low price of steviol glycosides and their derivatives and the ease of large - scale extraction and preparation, they have been widely used in the fields of food and beverage, medicine and organic chemistry, etc.

[0003] All SGs biosynthesis takes steviol as the precursor. Under the catalysis of different UGTs, different numbers of glucose, rhamnose and xylose groups are respectively linked to the C13 and C19 positions to form various SGs with different properties. Currently, 4 UGT - encoding genes involved in SGs biosynthesis are known, namely SrUGT85C2, SrUGT91D2, SrUGT74G1 and SrUGT76G1, and these genes can all participate in the glucosylation process of steviol glycosides. Among them, SrUGT85C2 can glycosylate the C13 - hydroxyl group of steviol; SrUGT74G1 glycosylates the C19 - carboxyl group of the steviol skeleton; SrUGT91D2 is responsible for catalyzing the formation of β - 1,2 glycosidic bonds between glucose; SrUGT76G1 is responsible for catalyzing the formation of β - 1,3 glycosidic bonds between glucose. These UGTs play roles successively and interact with each other in the SGs biosynthesis pathway, forming a complex glycosylation network, which makes Stevia rebaudiana contain more than 60 kinds of steviol glycosides, bringing great interference to the preparation and separation of glycosides.

[0004] In addition, although most of the genes in the SGs synthesis pathway have been successfully cloned and the related technologies have been relatively mature, the current research on the glycosylation pathway downstream of SGs synthesis is still very lacking, and many related problems have not been solved yet. For example, the biosynthesis process of xylosylated SGs has not been elucidated, and the flavor characteristics of most xylosylated SGs are not clear.

[0005] Among the currently known steviol glycosides, stevioside (STV) and rebaudioside A (Reb A) are the two main glycoside components. Among them, STV accounts for 5% - 10% of the dry leaf weight of the leaves, and its sweetness is 110 - 270 times that of sucrose; Reb A accounts for 2% - 4% of the dry leaf weight of the leaves, and its sweetness is 180 - 300 times that of sucrose. Currently, Reb A is one of the most common SGs used as a natural sweetener in the market, mainly extracted from stevia leaves. However, due to its slight bitter and minty taste, its development and application have been restricted.

[0006] Currently, it is known that the extension of the glycosyl unit linked at the C19 position can improve its sweetness. Rebaudioside D (Reb D) is obtained by 1,2-glycosylation of Reb A, with an extension of a glucose unit at the C19 position, and has a higher sweetness than Reb A. At the same time, linking different glycosyl groups will also affect the taste of SGs. Replacing the 1,2-glucosyl unit at the C19 position of Reb D with a 1,2-xylosyl unit produces rebaudioside FX1 (Reb FX1), which has a similar sweetness to Reb D and a lower bitter aftertaste, and has potential application prospects in the application of sweeteners. Therefore, it is of great significance to discover and identify glycosyltransferases involved in the synthesis of xylosylated SGs and with high C19 glycosylation selectivity from stevia for realizing the enzymatic synthesis and product separation of xylosylated SGs in vitro and promoting the research and application of the flavor characteristics of xylosylated SGs.

[0007] There have been many articles reporting O-xylosyltransferases in plants with secondary metabolites as acceptor substrates, but none of the currently reported UGTs in stevia have been shown to have xylosyltransferase activity, and the UGTs involved in the biosynthesis of xylosylated SGs have not been reported either. Moreover, although SrUGT91D2 has 1,2-glycosylation function, it is more inclined to glycosylation at the C13 position and cannot catalyze the formation of Reb D with a low sweetness threshold by 1,2-glycosylation of Reb A in vivo and in vitro, restricting its application. Summary of the Invention

[0008] The object of the present invention is to provide a glycosyltransferase gene with selectivity at the C19 position of steviol glycosides and the protein encoded thereby. The novel Stevia UGT provided by the present invention can specifically link a xylose to the glucose at the C19 position of steviol glycosides through a β-1,2 glycosidic bond. The change in the types of linked glycosyl groups effectively promotes the synthesis and separation of new SGs products. Moreover, the steviol glycoside β-1,2 glycosyltransferase with selectivity at the C19 position provided by the present invention differs from SrUGT91D2, which also has a 1,2-glycosylation function, in the way of substrate recognition and entry into the catalytic cavity, providing a theoretical and experimental basis for analyzing the substrate selection mechanism of glycosyltransferases involved in SGs synthesis. Overexpressing the glycosyltransferase of the present invention in Stevia rebaudiana provides a new direction for the variety improvement of Stevia rebaudiana.

[0009] The present invention provides a steviol glycoside glycosyltransferase, and its amino acid sequence has one or two of the following characteristics:

[0010] 1) It has the amino acid sequence shown in SEQ ID NO.1;

[0011] MDDHKQLHVAMFPWLAFGHILPFFELSKFITKNGHKVSFLSPTGNIQRLPSSNLSPLMNLVKLTLPRVQELPQNASATTDLHADDVQYLKQAFDGLQPEVTRFLEQESPDWIIYDFAPYWLPAVATSLGISRGFFSIFNAWTVSFFGSSPDDIINGTDDRKTADDFLTPPKWFPFPSKVCYRKHEANLIFADNISVNSSGVSDLYRLGMVIKGSDCMFIRHCHEFEPQWLTLLEKLHQLPVVPVGLLPPEPPTSTGDPWVTIKKWLDGQPIGHVVYVAFGSEGTMSQSELAELALGLELSGLPFFWVLRKPVGSGNSVELPEGFLERTRDRGLVWTSWVPQLQILSHESVCGFLTHSGWSSFVEAMMFGHPLIMLPLSVDQGLNARVMADNQVGIEIPRNDEDGSFTKESVARSLRLVLVDDEGKIYKAKAMELSQRFGDSKPENKYINPFIDYLEQKGRVVAIEHEL

[0012] 2) It has the amino acid residue sequence from the amino terminus of SEQ ID NO.1 at positions 1-468;

[0013] 3) An amino acid sequence with steviol glycoside glycosyltransferase activity formed by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1.

[0014] 4) An amino acid sequence that has at least 80% homology with SEQ ID NO.1 and has steviol glycoside glycosyltransferase activity.

[0015] The present invention also provides a steviol glycoside glycosyltransferase gene encoding the above steviol glycoside glycosyltransferase.

[0016] In the above technical solution, further, its nucleotide sequence has one or more of the following characteristics:

[0017] 1) Having the deoxyribonucleic acid sequence shown in SEQ ID NO.2;

[0018]

[0019] 2) A deoxyribonucleic acid sequence encoding the amino acid sequence of SEQ ID NO.1;

[0020] 3) A nucleotide sequence encoding a steviol glycoside glycosyltransferase activity obtained by substituting, deleting or adding one or more nucleotides to the deoxyribonucleic acid sequence of SEQ ID NO.2;

[0021] 4) A deoxyribonucleic acid sequence having a homology of 80% or more with the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.2 and capable of encoding a steviol glycoside glycosyltransferase.

[0022] The present invention also provides a recombinant expression plasmid containing the aforementioned steviol glycoside glycosyltransferase gene.

[0023] The present invention also provides a recombinant genetically engineered bacterium containing the aforementioned steviol glycoside glycosyltransferase gene.

[0024] The present invention also provides a method for preparing steviol glycoside glycosyltransferase, which clones the aforementioned steviol glycoside glycosyltransferase gene into a recombinant expression vector, introduces it into a host cell, and obtains a recombinantly expressed steviol glycoside glycosyltransferase;

[0025] Preferably, the expression vector for recombinantly expressing steviol glycoside glycosyltransferase is one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacterium expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0026] In the above technical solution, further, the host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacterium host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, or a mammalian cell.

[0027] The present invention also provides the application of the aforementioned steviol glycoside glycosyltransferase in the production of steviol glycosides.

[0028] In the above technical solution, further, the steviol glycoside product is a steviol glycoside compound having a β-1,2 xylose sugar chain at the C19 position; preferably, the steviol glycoside is a 19-xylosylated derivative of rubusoside, a 19-xylosylated derivative of stevioside, or rebaudioside FX1.

[0029] The present invention also provides the application of the aforementioned steviol glycoside glycosyltransferase gene in the genetic breeding of Stevia rebaudiana.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention discloses for the first time a xylosyltransferase-encoding gene SrUGT91D6 with steviol glycoside C19 position selectivity and the protein encoded thereby. SrUGT91D6 has strong C19 glycosylation selectivity, has high activity towards substrates with C13 and C19 disaccharide glycosylation sites, and has weak activity towards substrates with only C19 glycosylation sites. Compared with the glycosyltransferases identified in stevia currently, the β-1,2-glycosyltransferase with C19 position selectivity provided by the present invention can effectively prepare and isolate new xylosylated steviol glycosides, and can achieve in vitro enzymatic synthesis of Reb FX1 with high sweetness and low bitterness. The β-1,2-glycosyltransferase with C19 position selectivity provided by the present invention has changed in the way of substrate recognition and substrate entry into the catalytic cavity compared with SrUGT91D2, providing a theoretical and experimental basis for the analysis of the substrate selection mechanism of β-1,2-glycosyltransferase. Overexpression of the glycosyltransferase of the present invention in stevia provides a new direction for the improvement of stevia varieties. Description of the Drawings

[0031] Figure 1 : Sequence alignment result diagram of SrUGT91D6 protein sequence in NCBI database;

[0032] Figure 2 : Glycosyltransferase family classification of target gene SrUGT91D6;

[0033] Figure 3 : Three-dimensional structure alignment diagram of SrUGT91D6 protein and OsUGT91C1;

[0034] Figure 4 : Electrophoresis diagram of ORF amplification product of target gene SrUGT91D6;

[0035] Figure 5 : SDS-PAGE electrophoresis diagram of SrUGT91D6 protein.

[0036] Wherein: M: Protein molecular weight standard; Lane 1: Purification of SrUGT91D6 protein.

[0037] Figure 6 : Liquid phase and mass spectrometry detection diagram of in vitro catalysis of rubusoside by SrUGT91D6.

[0038] Figure 7 : Liquid phase and mass spectrometry detection diagram of in vitro catalysis of stevioside by SrUGT91D6.

[0039] Figure 8: In vitro catalytic liquid phase and mass spectrometry detection diagrams of SrUGT91D6 for rebaudioside A.

[0040] Figure 9 : In vitro catalytic mass spectrometry detection diagrams of SrUGT91D6 for rubusoside.

[0041] Figure 10 : Comparison of the conversion rates of SrUGT91D6 using rubusoside, stevioside, and rebaudioside A as substrates.

[0042] Figure 11 : Sequence alignment diagrams of SrUGT91D6 and similar sequences.

[0043] Figure 12 : In vitro catalytic liquid phase diagrams of SrUGT91D6 and its similar sequences SrUGT91D6-1 / 2 / 3 for rubusoside.

[0044] Figure 13 : In vitro catalytic liquid phase diagrams of SrUGT91D6 for the flavonoid substrate luteolin.

[0045] Figure 14 : Glycosylation roadmap of steviol glycosides by SrUGT91D6. Detailed implementation methods

[0046] The present invention will be described in detail below with reference to examples. The examples are for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following implementation methods are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0047] Example 1 Transcriptome sequencing of leaves of different Stevia rebaudiana varieties

[0048] Using different Stevia rebaudiana varieties rich in different types of SGs as experimental materials, collect the middle leaves of Stevia rebaudiana plants after 2 months of growth, and store them at -80 °C for later use. Extract RNA and perform detection, and use the Illumina HiSeq TM 2000 sequencing platform for transcriptome library sequencing.

[0049] Example 2 Screening of UGTs genes based on Stevia rebaudiana transcriptome data

[0050] Using the three known steviol glycoside-1,2-UGTs as queries, namely OsUGT91C1, LbUGT, and UGTSL2, perform a blast in the transcriptome database and conduct further analysis and screening on the output results. Obtain the FPKM values of each glycosyltransferase gene in different varieties of Stevia rebaudiana using the transcriptome data. Construct a phylogenetic tree using MEGA 7.0 to analyze the homology of the candidate glycosyltransferase genes. Use Alphafold to construct a model of the candidate glycosyltransferase, with the crystal structure of OsUGT91C1 as a template, and in situ simulate the binding of the candidate glycosyltransferase to SGs.

[0051] Analysis of the phylogenetic tree revealed that sequences with high homology to LbUGT and UGTSL2 had no expression levels, while SrUGT91D6 had high homology with OsUGT91C1 and SrUGT91D2 ( Figure 2 ). Among all the sequences belonging to the UGT91 family, SrUGT91D6 had relatively high expression levels in different varieties of Stevia rebaudiana. Aligning the sequence of SrUGT91D6 with the currently known SrUGT91D2 that can catalyze the 1,2-glycosylation of SGs, it was found that their similarity was only 65.53% ( Figure 1 ). The three-dimensional structure of SrUGT91D6 had the highest similarity with OsUGT91C1, which was 39.15%. OsUGT91C1 can use SGs as the acceptor substrate to extend the sugar chain through 1,2-glucosylation ( Figure 3 ). Structure alignment and docking analysis showed that the substrate cavity sizes of SrUGT91D6 and OsUGT91C1 were similar and could accommodate SGs molecules as substrates. Therefore, it was inferred that SrUGT91D6 might be a steviol glycoside 1,2-glycosyltransferase in Stevia rebaudiana.

[0052] Example 3 Cloning and Expression of the SrUGT91D6 Encoding Gene

[0053] Design primers based on the full-length sequence of SrUGT91D6 obtained from the transcriptome sequencing and amplify the target gene from Stevia rebaudiana cDNA ( Figure 4 ).

[0054] Full-length primers:

[0055] SrUGT91D6-F:

[0056] CAGCAAATGGGTCGCGGATCCATGGACGACCATAAGCAGCTTCA

[0057] (SEQ ID NO.3)

[0058] SrUGT91D6-R:

[0059] TGGTGGTGCTCGAGTGCGGCCGCAAAGCTCATGCTCAATAGCAACCAC

[0060] (SEQ ID NO.4)

[0061] After sequencing verified the sequence correctness, the target gene was ligated to the pET28a vector digested with BamHI and NotI using the Gibson Assembly method. Take 10 μL of the ligation product and transform it into E. coli TOP10 competent cells, then spread it on solid Luria-Bertani medium containing 100 μg / mL kanamycin and culture at 37 °C for 12 - 16 h. Pick monoclonal colonies, verify them by colony PCR using universal primers, inoculate the correctly amplified monoclonal colonies into liquid Luria-Bertani medium containing 100 μg / mL kanamycin for culture, and extract plasmids; use the restriction enzymes BamHI and NotI to double digest the extracted plasmids, and send the recombinant plasmids with correct results for sequencing at BGI. The sequencing results showed that the candidate gene shown in SEQ ID NO 2 was inserted between the BamHI and NotI restriction sites of pET28a, and the insertion direction was correct, proving that the recombinant plasmid was successfully constructed, and these recombinant plasmids were named pET28a-SrUGT91D6. The full nucleotide sequence of the steviol glycoside glucosyltransferase SrUGT91D6 gene is 1404 bp in length, and the nucleotide sequence is as shown in SEQ ID NO.2; it encodes 468 amino acids, and the amino acid sequence is as shown in SEQ ID NO.1. The theoretical molecular weight of the protein is 52.6 kDa( Figure 5 ).

[0062] Transform pET28a-SrUGT91D6 into E. coli BL21(DE3), and perform its induction expression and purification. Detect the expression and purification of SrUGT91D6 by polyacrylamide gel electrophoresis. The purified glucosyltransferase showed a single band on the electrophoresis gel, and its position was consistent with the predicted molecular weight.

[0063] Example 4 Detection of SrUGT91D6 Glycoside Substrates

[0064] The purified glucosyltransferase protein was functionally verified in vitro according to the following reaction system (100 μL):

[0065] 20 mM Tris (pH 8.0), 100 mM sodium chloride, 70 μg of purified protein, 0.5 mM UDP-Xylose, 0.5 mM of receptor substrates, and the receptor substrates include: Rubusoside, STV, Reb A. After reacting at 37 °C for 18 hours, an equal volume of absolute ethanol was added to terminate the reaction, shaken and mixed well, centrifuged at 12,000 rpm for 1 minute, the supernatant was taken and filtered through a 0.22 μm organic filter membrane, and the composition of the reaction products was qualitatively and quantitatively detected by liquid chromatography. Instrument model, Waters e2695. Injection volume 20 μL, chromatographic column: Elite superil ODS2 (5 μm, 250×4.6 mm), column temperature: 40 °C. Chromatographic conditions: UV 210 nm, mobile phase: (A): water, (B): acetonitrile, flow rate: 1 mL / min, elution program: 0 - 2 min, 20% B; 2 - 10 min, linearly increasing B phase to 33.5% B; 10 - 13 min, 33.5% B; 13 - 16.5 min, 33% B; 16.5 - 26 min, linearly increasing to 80% B; 26 - 30 min, 20% B.

[0066] The results of liquid chromatography detection are as Figures 6 - 8 shown. SrUGT91D6 can glycosylate Rubusoside, STV, and Reb A. Since only the glycosyl group linked at the C19 position of STV and Reb A can undergo 1,2-glycosylation, it indicates that this enzyme has the ability to glycosylate the glycosyl group at the C19 position. Both the C13 and C19 positions of Rubusoside can undergo 1,2-glycosylation. Further LC-MS / MS analysis was performed on the product of SrUGT91D6 glycosylating Rubusoside, and the results showed ( Figure 9 ) that the fragment ions of the C19-glycosylated product of Rubusoside were the main fragment ion peaks (the mass-to-charge ratio of the fragment ions was approximately 479), and only a small amount of fragment ion peaks of the C13-glycosylated product were contained (the mass-to-charge ratio of the fragment ions was approximately 611), indicating that SrUGT91D6 has specific C19-glycosylation ability. SrUGT91D2 glycosylating Rubusoside will produce STV, cannot glycosylate Reb A to generate Reb D, and can almost only perform 1,2-glycosylation at C13, lacking the ability of C19-glycosylation. While SrUGT91D6 has specific C19-glycosylation function, can glycosylate Reb A to generate Reb FX1 with a better taste, and promotes the synthesis of high-sweetness glycosides in vitro. SrUGT91D6 has a relatively high glycosylation ability for Rubusoside with only one glycosyl group linked at the C13 position, and as the number of glycosyl groups linked at C13 increases, its activity towards STV and Reb A decreases ( Figure 10), so it is speculated that in the complex steviol glycoside mixture system or in Stevia rebaudiana, SrUGT91D6 will be beneficial to promoting the synthesis of structurally diverse steviol glycosides( Figure 14 ), enriching the types of steviol glycosides and providing more possibilities for the application of steviol glycosides.

[0067] Example 5 Exploration of SrUGT91D6 gene polymorphism in different varieties of Stevia rebaudiana

[0068] By amplifying the target gene from the cDNA of different varieties of Stevia rebaudiana, other sequences with extremely high similarity were obtained in addition to SrUGT91D6( Figure 11 ), namely SrUGT91D6-1 (SEQ ID NO.5 is the amino acid sequence, Identity = 98.29%; SEQ ID NO.8 is the nucleotide sequence), SrUGT91D6-2 (SEQ ID NO.6, Identity = 99.79%; SEQ ID NO.9 is the nucleotide sequence), SrUGT91D6-3 (SEQ ID NO.7, Identity = 98.93%; SEQ ID NO.10 is the nucleotide sequence). And they were verified in subsequent enzyme activity detection experiments, and the results showed that these enzymes have the same function as SrUGT91D6( Figure 12 ).

[0069] Example 6 Detection of SrUGT91D6 flavonoid substrates

[0070] The purified glycosyltransferase protein was functionally verified in vitro according to the following reaction system (100 μL):

[0071] 20 mM Tris (pH 8.0), 100 mM sodium chloride, 100 μg of purified protein, 0.5 mM UDP-Glucose or UDP-Xylose, 0.5 mM of the acceptor substrate, and the acceptor substrate is luteolin. After reacting at 37 °C for 18 hours, an equal volume of absolute ethanol was added to terminate the reaction, shaken and mixed evenly, centrifuged at 12000 rpm for 1 minute, the supernatant was taken and filtered through a 0.22 μm organic filter membrane, and the composition of the reaction products was qualitatively and quantitatively detected by liquid chromatography. Instrument model, Waters e2695. Injection volume 10 μL, chromatographic column: Elite superil AQ-C18 (5 μm, 250×4.6 mm), column temperature: 40 °C. Chromatographic conditions: UV 210 nm - 400 nm, mobile phase: (A): 0.1% formic acid, (B): methanol, flow rate: 1 mL / min, elution program: 0 - 3 min, 20% B; 3 - 18 min, linearly increasing B phase to 60% B; 18 - 25 min, linearly increasing to 100% B.

[0072] Liquid chromatography detection results(Figure 13 ), SrUGT91D6 can catalyze the xylosylation of luteolin to generate the corresponding product, but cannot catalyze the glucosylation of luteolin. This indicates that SrUGT91D6 can not only use steviol glycosides as the acceptor substrate, but also use flavonoids as the acceptor substrate, with a broad substrate spectrum. At the same time, when using luteolin as the substrate, SrUGT91D6 also prefers to use UDP-xylose, indicating its preference for UDP-xylose.

[0073] For any person skilled in the art, without departing from the scope of the technical solution of the present invention, many possible changes and modifications can be made to the technical solution of the present invention by using the technical content disclosed above, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A steviol glycoside glycosyltransferase, characterized in that, Its amino acid sequence is as shown in SEQ ID NO.1; or an amino acid sequence having at least 80% homology with SEQ ID NO.1 and having steviol glycoside glycosyltransferase activity.

2. A steviol glycoside glycosyltransferase gene, characterized in that, Encoding the steviol glycoside glycosyltransferase described in claim 1.

3. The steviol glycoside glycosyltransferase gene according to claim 2, characterized in that, Its nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO.1; Or having a homology of 80% or more with the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.2 and being able to encode the deoxyribonucleic acid sequence of the steviol glycoside glycosyltransferase of claim 1.

4. A recombinant expression plasmid containing the steviol glycoside glycosyltransferase gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium containing the steviol glycoside glycosyltransferase gene according to claim 2 or 3.

6. A method for preparing steviol glycoside glycosyltransferase, characterized in that: Cloning the steviol glycoside glycosyltransferase gene described in claim 2 or 3 into a recombinant expression vector, introducing it into a host cell, and obtaining a recombinantly expressed steviol glycoside glycosyltransferase; Preferably, the expression vector for the recombinantly expressed steviol glycoside glycosyltransferase includes one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

7. The method according to claim 6, characterized in that: The host cell includes an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell, a mammalian cell.

8. The application of the steviol glycoside glycosyltransferase according to claim 1 in the production of xylosyl-containing steviol glycosides.

9. The application according to claim 8, characterized in that, The steviol glycoside is a steviol glycoside product having 1,2-xylosylation at the C19 or C13 position; preferably, the steviol glycoside product is a 13- or 19-position xylosylation derivative of rubusoside, a 19-position xylosylation derivative of stevioside, rebaudioside FX1.

10. The application of the steviol glycoside glycosyltransferase gene according to claim 2 or 3 in the genetic breeding of Stevia rebaudiana.

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