CN112695070B

CN112695070B - Novel method for measuring glycosyltransferase activity

Info

Publication number: CN112695070B
Application number: CN202110002225.0A
Authority: CN
Inventors: 吴旭日; 李萌; 夏媛; 杜雅丽; 赵玲; 陈依军
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-01-04
Filing date: 2021-01-04
Publication date: 2023-08-01
Anticipated expiration: 2041-01-04
Also published as: CN112695070A

Abstract

The invention belongs to the technical field of biocatalysis, and particularly relates to a novel method for measuring glycosyltransferase activity, which is characterized in that fructose generated in a double-enzyme reaction system of UDP-glucose dependent glycosyltransferase coupled sucrose synthase is measured by a chromogenic method so as to characterize the glycosyltransferase activity. The invention discloses a novel color reaction-based UDP-glucose dependent glycosyltransferase screening method, which provides a preliminary activity screening tool for the transformation and the development of glycosyltransferase and also provides a novel method for the activity characterization of UDP-glucose dependent glycosyltransferase.

Description

Novel method for measuring glycosyltransferase activity

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a novel method for detecting and screening UDP-glucose dependent glycosyltransferase activity based on color reaction.

Background

Glycosyltransferase plays a variety of metabolism and regulation roles by catalyzing the transfer of sugar molecules to different receptor substrates, and is widely applied to the fields of research of disease treatment targets, drug development and the like. UDP-glucose dependent glycosyltransferase (UGTs) takes uridine diphosphate glucose (UDPG) as glycosyl donor, can selectively catalyze natural products and non-natural compounds to synthesize glycosylation products with different biological activities and physicochemical properties, and is one of important tool enzymes for developing medicines and food additives. With the continued depth of UGT research and the increasing difficulty of new enzyme mining, the use of wild-type UGTs with weak catalytic activity and narrow substrate spectrum in glycosylation modification of natural and non-natural compounds is limited (Li C, et al trends Biotechnol.2020, 38:729-744). At present, random mutation and directed evolution are important strategies for improving the catalytic properties of UGTs and designing artificial new enzymes, however, development of efficient screening methods capable of characterizing the activities of UGTs and mutants thereof has been one of technical difficulties in preventing UGTs from being transformed.

To solve the above problems, researchers have developed some UGTs screening methods, but all have disadvantages that are difficult to apply effectively: (1) Radiolabelling is highly sensitive, but is complex to operate and produces radiochemicals (Brown C, et al Nat Protoc,2012, 7:1634-1650); (2) Immunological methods are also very sensitive, but limited by the cost of detection and stability of antibody binding to the receptor (Chokhawala HA, et al ACS Chem Biol,2008, 3:567-576); (3) Chromatography and mass spectrometry have obvious advantages in accuracy, but are costly and time consuming and cannot be performed in high throughput (Kopp M, et al Chembiochem,2007,8:813-819; gurrad-Levin ZA, et al ACS combSci, 2011, 13:347-350); (4) Screening methods based on pH indicators are less sensitive and specific and have a large number of interfering factors (Deng C, et al Anal Biochem,2004, 330:219-226); (5) Fluorescence-based screening methods require the preparation of complex structures and the fluorescent signal is susceptible to interference (Lee HS, et al, animal Biochem,2011,418:85-88;Ryu J,et al.Bioorg Med Chem,2014,22:2571-2575); (6) Spectrophotometry combines UDP or glycosylation products from glycosylation with other enzymatic reactions, which are relatively complex to operate and are only applicable to pure enzymes (Li Y, et al J Biotechnol,201, 227:10-18). Therefore, the method for determining the activity of UDP-glucose dependent glycosyltransferase is simple, quick, low in cost and high in flux, and has important application value for the development and modification of glycosyltransferase.

Disclosure of Invention

Object of the Invention

UDP glucose dependent glycosyltransferase is an important tool enzyme and has great application prospect in the aspects of compound structural modification and pharmaceutical modification. However, mutant screening and new enzyme mining in the process of modifying UDP glucose-dependent glycosyltransferase involve a large number of screening works, and the existing screening and activity detection methods have certain defects, so that in order to overcome the defects, the invention provides a new method for measuring and screening the glycosyltransferase activity based on color reaction, and aims to provide a feasible screening tool.

Technical proposal

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a new method for measuring glycosyltransferase activity, which is characterized in that fructose generated in a double-enzyme reaction system of UDP-glucose dependent glycosyltransferase coupled sucrose synthase is measured based on a chromogenic method to characterize the glycosyltransferase activity.

The novel method for measuring the glycosyltransferase activity is characterized in that the UDP-glucose dependent glycosyltransferase and the sucrose synthase can be pure enzyme or crude enzyme extract.

The novel method for determining the glycosyltransferase activity is characterized in that the UDP-glucose dependent glycosyltransferase and the sucrose synthase can be expressed in escherichia coli, streptomycete, saccharomycete, bacillus subtilis, bacteriophage and CHO cell expression systems singly or together.

The novel method for measuring the glycosyltransferase activity is characterized in that the escherichia coli is an engineering strain E.coli MalZ-KO of which the maltodextrin glucosidase coding gene malZ is knocked out by E.coli BL21 (DE 3).

The novel method for measuring the glycosyltransferase activity is characterized in that the color reaction is DNS reaction, filin reagent reaction, benedict reagent reaction and barsurface reagent reaction.

The novel method for glycosyltransferase activity is characterized in that the method can be used for detecting the activity of UDP-glucose dependent glycosyltransferase, screening novel UDP-glucose dependent glycosyltransferase and screening the mutated activity of UDP-glucose dependent glycosyltransferase.

The novel method for measuring the glycosyltransferase activity is characterized in that UDP-glucose dependent glycosyltransferase is Oled-ASP, and the amino acid sequence of the UDP-glucose dependent glycosyltransferase is SEQ ID NO. 2; the sucrose synthase is SUS1, and the amino acid sequence is SEQ ID NO. 5.

Specifically

1) And determining alpha-glucosidase MalZ of fructose generated by hydrolyzing sucrose in E.coli BL21 through KEGG analysis, knocking out to construct an engineering strain E.coli MalZ-KO, reducing detection background interference, and providing a chassis strain for the development of the method.

2) Oled-ASP and sucrose synthase are respectively expressed or co-expressed in E.coli malZ-KO to construct a double-enzyme coupling reaction system, and the feasibility of the activity determination method of UDP-glucose dependent glycosyltransferase based on color reaction is verified.

3) Optimizing the catalytic reaction conditions of Oled-ASP and sucrose synthase creates a new method for determining the activity of UDP-glucose dependent glycosyltransferase based on color reaction.

4) The inventor earlier application patent (application number: 202010921312.1) the UDP-glucosyltransferase GT-2, having the amino acid sequence SEQ ID NO 3, and GT-5, having the amino acid sequence SEQ ID NO 4, coupled with sucrose synthase catalyzes the glycosylation modification of the nosiheptide as a model reaction to verify the utility of the method.

Wherein:

1. KEGG (Kyoto Encyclopedia of Genes and Genomes, kyoto gene and genome encyclopedia) is a comprehensive database of systematic analysis of gene functions, a knowledge base of contact genomic and functional information. KEGG Pathway, one of its cores, is a metabolic Pathway diagram hand-drawn according to related knowledge, and is divided into 6 classes, metapolism, genetic information Processing, cellular Processes, environmental information Processing, organismal Systems, human Diseases, respectively. These KEGG paths contain information on the number of proteins, compounds, and interactions between them.

In the KEGG metabolic pathway database (KEGG Pathway Database), starch and sucrose metabolism (00500 Starch and sucrose metabolism) was selected under carbohydrate metabolism (1.1 Carbohydrate metabolism).

According to analysis, in the sucrose metabolic pathway of E.coli BL21 (DE 3), maltodextrin glucosidase (MalZ) can catalyze sucrose to generate fructose and glucose, which possibly generates background interference on the screening method based on detection of fructose in the invention, so that the MalZ gene is knocked out.

2. Principle of DNS (dinitrosalicylic acid) color development;

the reducing sugar can be oxidized to sugar acids and other products by heating under alkaline conditions, while the oxidant 3, 5-dinitrosalicylic acid is reduced to reddish-brown 3-amino-5-nitrosalicylic acid. In a certain range, the amount of the reducing sugar is in direct proportion to the color depth of the brown red substance, the optical density value is measured at the wavelength of 540nm by utilizing a spectrophotometer, and the content of the reducing sugar and the total sugar in the sample can be obtained by checking and calculating a standard curve.

Advantageous effects

1. The invention discloses a novel color reaction-based UDP-glucose dependent glycosyltransferase activity detection or screening method, which belongs to the first report. The general flow is as follows: constructing a coupling reaction system of UDP-glucose dependent glycosyltransferase and sucrose synthase, and detecting fructose generated by double-enzyme coupling reaction by using a DNS chromogenic method, thereby establishing an activity detection or screening method of the glycosyltransferase based on color reaction. In order to achieve the above object, the invention uses UDP-glucose dependent glycosyltransferase Oled-ASP and sucrose synthase to catalyze the glycosylation modification of 4-methylumbelliferone in a coupled mode to carry out the construction of the method, and uses glycosyltransferase to catalyze the reaction for synthesizing glycosylated norcetin to verify the applicability of the method.

2. The color reaction-based UDP-glucose dependent glycosyltransferase rapid activity determination method has the advantages of simple operation, low cost, high flux and the like, can be used for determining the activity of UDP-glucose dependent glycosyltransferase, screening novel UDP-glucose dependent glycosyltransferase, screening the mutation activity of UDP-glucose dependent glycosyltransferase and the like, and provides a preliminary screening tool for the development and reconstruction of glycosyltransferase.

Drawings

FIG. 1 is a SDS-PAGE analysis of independent expression of sucrose synthase (A) and Oled-ASP (B);

FIG. 2 is a fumbling of a method for determining the activity of UDP-glucose dependent glycosyltransferase based on a color reaction;

FIG. 3 is a SDS-PAGE analysis of sucrose synthase (A) and Oled-ASP co-expression, wherein M: protein molecular weight standard; lane 1: e.coli BL21 (DE 3) control; lane 2: e.coli malZ-KO control; lanes 3,4: e.coli K &1; lanes 5-6: e.coli K &2;

FIG. 4 is a comparison of the reaction efficiencies of E.coli K &1 (A) and E.coli K &2 (B);

FIG. 5 is a schematic representation of plasmid pETDuet-1-1ATSUS 1-oleD-Asp;

FIG. 6 is a schematic diagram of a screening according to the present invention.

Detailed Description

The specific steps of the present invention are described below by way of examples, but the scope of the present invention is not limited by this example.

Preparing a DNS reagent: 18.2g of potassium sodium tartrate is dissolved in 50ml of distilled water, heated, 0.63g of 3, 5-dinitrosalicylic acid, 0.5g of NaOH2.1g of phenol are sequentially added into the hot solution, stirred until the solution is dissolved, cooled, then distilled water is used for constant volume to 100ml, and the solution is stored in a brown bottle at room temperature.

EXAMPLE 1 HPLC analysis conditions

Chromatographic column: YMC-Pack ODS-A (150X 4.6mm,5 μm,12 nm); mobile phase a: ultrapure water (containing 0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid); sample injection volume: 2. Mu.L; flow rate of system: 1.0mL/min; column temperature: 30 ℃; detection wavelength: 232nm; elution gradient: 10 to 50 percent of B is eluted linearly for 6min, and 50 to 70 percent of B is eluted linearly for 16min.

Example 2 DNS color method

Preparing fructose solutions with different concentrations (see table 1), taking 0.3mL of each fructose solution with each concentration, sequentially adding 0.3mL of DNS reagent, fully mixing, and centrifuging at 12000rpm for 30s; centrifuging in boiling water bath at 12000rpm for 30s, cooling to room temperature, and measuring OD ₅₄₀ Values according to fructose concentration and corresponding OD ₅₄₀ Values are plotted as fructose standard curves. The standard curve equation is y= 0.4497x-0.0345 (r ² =0.999), where y is OD _540nm X is the fructose concentration.

TABLE 1 preparation of fructose solutions

After the double-enzyme coupling reaction is finished, the reaction solution is subjected to water bath at 80 ℃ for 5min to terminate the reaction, 0.3mL of supernatant is taken after centrifugation, 0.3mL of DNS reagent is added, and after full mixing, the mixture is centrifuged at 12000rpm for 30s; centrifuging in boiling water bath at 12000rpm for 30s, cooling to room temperature, and measuring OD _540nm Values. In the measurement, the concentration is required to be too highFixed liquid is diluted to OD _540nm To within 1.0.

EXAMPLE 3λRed Gene recombination knockout of malZ Gene

In the KEGG metabolic pathway database (KEGG Pathway Database), the starch and sucrose metabolism (00500 Starch and sucrose metabolism) module under carbohydrate metabolism (1.1 Carbohydrate metabolism) is selected, the strain E.coli BL21 (DE 3) to be analyzed is selected, the starch and sucrose metabolic pathway map thereof (https:// www.kegg.jp/KEGG-bin/show_pathwayebl 00500) is obtained, and the sucrose catabolic pathway is analyzed accordingly, and the key enzyme alpha-glucosidase MalZ (SEQ ID NO: 1) for sucrose hydrolysis is determined.

50bp upstream and downstream of maltodextrin glucosidase gene malZ is selected as a homology arm, and then added to 5' ends of pKD3 plasmid universal amplification primers P1 and P2 respectively, and finally primers malZ-homo-F (SEQ ID NO: 11) and malZ-homo-R (SEQ ID NO: 12) for homologous substitution fragment amplification are obtained.

malZ-homo-F：

5’-TGCATTAGGCTATGGCAAGGTGATCAGATTTTCATCACAGGGGAATTATGGTGTAGGCTGGAGCTGCTTC-3’

malZ-homo-R：

5’-GTTTTATCCGCGGATGATGGCGCAGGCGTCACGCAAGGCGTTATAAAACGATGGGAATTAGCCATGGTCC-3’

The pKD3 plasmid is used as a template to amplify a substitution fragment with a homology arm (the fragment contains FRT site and chloramphenicol resistance gene), and after the PCR reaction is finished, the PCR amplified fragment is separated by 1% agarose gel electrophoresis, and the homologous substitution fragment is recovered.

The pKD46 plasmid is transformed into E.coli BL21 competence, homologous substitution fragment is added into E.coli BL21 electrotransformation competence to carry out homologous recombination, then the pKD46 plasmid in positive recombinant bacteria is lost by culturing at 42 ℃, then the pCP20 plasmid is transformed into recombinant bacteria competence, the plasmid acts on FRT site to eliminate chloramphenicol resistance of the bacteria, and the plasmid is lost by culturing at 42 ℃. Finally obtaining the E.coli malZ-KO strain.

EXAMPLE 4E background detection of collZ-KO

Ultrasonic disruption of E.coll malZ-KO and E.coll BL21, and centrifugation to obtain supernatant. According to the total volume of the reaction system being 1mL, the final concentration of each component is as follows: DNS color reaction was performed with sucrose solution 0.2mol/L, crude enzyme solution 0.5mL, buffer 0.05mol/L phosphate buffer (pH 7.5). The result shows that the fructose concentration generated by E.coli BL21 is 2.326mmol/L, the fructose concentration generated by E.coli malZ-KO is 0.074mmol/L, the fructose concentration is reduced by 31.5 times, and the background interference of E.coli BL21 is completely eliminated.

Example 5 soluble expression and detection method of Oled-ASP and sucrose synthase SUS1

The recombinant plasmids pET22b-sus1, pET22b-Oled-ASP were transformed into E.coli malZ-KO and induced to express at 0.5mM IPTG and 20℃for 12h. As can be seen from SDS-PAGE, oled-ASP (SEQ ID NO: 2) and sucrose synthase SUS1 (SEQ ID NO: 5) were both normally expressed in a soluble manner (FIG. 1).

On the basis of the double enzyme soluble expression, a 5mL reaction system was constructed to verify the feasibility of the UDP-glucose activity assay method based on the color reaction. 5mL of the reaction system contained 1mg/mL of 4-methylumbelliferone, 3.5mg/mL of UDP-glucose, 0.2M of sucrose, 1.25mL of Oled-ASP crude enzyme solution, 1.25ml,2.5ml Tris-HCl buffer (pH 8.0, containing 5mM MgCl) ₂ ). The reaction is carried out for 12h at 30 ℃ and 220 r/min. As shown in FIG. 2, the double enzyme reaction group showed a clear orange color, OD, compared with the empty bacterial control of E.coli malZ-KO _540nm 1.37. The results indicate that a UDP-glucose activity assay based on DNS method for detecting fructose is feasible.

EXAMPLE 6 construction of Co-expression Strain

The co-expression vector pETDuet-1 is adopted, enzyme cutting sites such as Nco I/Hind III, nde I/Xho I and the like are used for constructing a co-expression plasmid pETDuet-1-1ATSUS1-oleD-Asp of sucrose synthase (Arabidopsis thaliana sucrose synthase is abbreviated as AtSUS 1) and Oled-ASP, and E.coli malZ-KO is introduced to obtain a co-expression strain E.coli K&1 and E.coli K&2 (table 2). The induction expression condition of the engineering strain IPTG is 0.4mM IPTG, the temperature is 20 ℃, the SDS-PAGE detects the expression quantity (figure 3), and then a 5mL reaction system is constructed: 1mg/ml of 4-methylumbelliferone, 3.5mg/ml of UDP-glucose, 0.2M of sucrose, 2.5ml of crude enzyme solution and 2.5ml Tris-HCl buffer (pH 8.0, containing 5mM MgCl2). The reaction is carried out for 12 hours at 30 ℃ and 220r/min, and then DNS color reaction is carried out. The results are shown in FIG. 4, E.coli K&1 and E.coli K&2 can all normally catalyze sucrose to produce fructose, and E.coli K&2 vitality (OD) _540nm =1.85) is slightly higher than e.coli K&1(OD _540nm =1.73), so select e.colli K&2 follow-up study was performed.

TABLE 2 Co-expression Strain

Example 7 optimization of double enzyme coupling reaction conditions

A double enzyme reaction system was constructed as in example 6, and the reaction temperature (25℃to 40 ℃) and the reaction pH (6.5 to 9.0) and the reaction time (2 hours to 14 hours) were examined for OD, respectively _540nm And the effect of 4-methylumbelliferone conversion. 1) Optimal reaction temperature: 30 ℃, OD _540nm The maximum value is 1.83. The conversion of 4-methylumbelliferone was 66.5%; 2) Optimal reaction pH: pH8.0, OD _540nm The maximum value is 1.83. The conversion of 4-methylumbelliferone was 67.5%; 2) Optimal reaction time: 10h, OD _540nm The maximum value is 2.36. The conversion of 4-methylumbelliferone was 82.8%. Thus, the optimal conditions for the double enzyme reaction were finally determined to be 30℃and pH8.0, and the reaction was continued for 10 hours, as shown in FIG. 6.

EXAMPLE 8 investigation of the detection limits of color reaction

Detection of OD of blank test group (without fructose) by DNS color development ₅₄₀ Values, repeat the assay 10 times. According to the fructose standard curve of example 2, the detection limit is calculated as follows:

LOD＝3s/b

( LOD: a detection limit; s: standard deviation of measured values; b: slope of linear regression equation )

The detection limit of the color reaction was calculated to be 0.026mmol/L.

EXAMPLE 9 investigation of color reaction stability

Fructose solutions (0.5 mmol +.L, 0.75mmol/L, 1.0mmol/L, 1.25mmol/L, 1.5 mmol/L), and then is placed in a room at room temperature to measure OD at 0min, 10min, 20min, 30min, 45min, 60min, respectively _540nm Values. The result shows that under the normal temperature condition, the color-developing substance 3-amino-5-nitro salicylic acid generated by the action of fructose solution with different concentrations and DNS reagent is placed indoors for 60min, and the OD thereof _540nm The values did not change. This indicates that the stability of the color reaction is better.

Example 10 application of color-responsive glycosyltransferase-based screening method

The glycosylation modification of the norcetirin is catalyzed by an enzyme method to be a model reaction so as to verify the practicability of the activity measurement method based on the color reaction. UDP-glucose dependent glycosyltransferases GT-2 and GT-5 of the inventor (application number: 202010921312.1) are selected as catalysts, corresponding co-expression plasmids are respectively constructed with sucrose synthase AtSUS1, and co-expression strains E.coll K &3 and E.coll K &4 are respectively obtained after E.coll malZ-KO is transformed. The conditions for induction expression of IPTG were 0.4mM IPTG at 20℃for 14 hours.

A5 mL reaction system was constructed as in example 6: noxipeptide 1mg/ml, UDP-glucose 3.5mg/ml, sucrose 0.2M, crude enzyme 2.5ml,2.5ml Tris-HCl buffer (pH 8.0, containing 5mM MgCl) ₂ ). The reaction was carried out at 30℃and 220r/min for 10 hours, followed by a DNS coloration reaction. The results show that E.coli K&OD of 4 reaction group _540nm 1.17, significantly higher than E.coli K&3 reaction group 0.52. The trend of the results is consistent with the trend of the enzyme activity of the prior invention patent (application number: 202010921312.1) of the inventor. In conclusion, the UDP-glucose dependent glycosyltransferase activity screening method based on the color reaction can be used for enzyme activity measurement, mutant activity screening and the like, and is an effective enzyme activity primary screening method.

Sequence listing

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Met Arg Leu Val Leu Asp Gly Val Phe Asn His Ser Gly Asp Ser His

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Ala Trp Phe Asp Arg His Asn Arg Gly Thr Gly Gly Ala Cys His Asn

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Pro Glu Ser Pro Trp Arg Asp Trp Tyr Ser Phe Ser Asp Asp Gly Thr

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Ala Leu Asp Trp Leu Gly Tyr Ala Ser Leu Pro Lys Leu Asp Tyr Gln

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Ser Glu Ser Leu Val Asn Glu Ile Tyr Arg Gly Glu Asp Ser Ile Val

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Arg His Trp Leu Lys Ala Pro Trp Ser Met Asp Gly Trp Arg Leu Asp

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Val Val His Met Leu Gly Glu Ala Gly Gly Ala Arg Asn Asn Met Gln

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His Val Ala Gly Ile Thr Glu Ala Ala Lys Glu Thr Gln Pro Glu Ala

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Tyr Ile Val Gly Glu His Phe Gly Asp Ala Arg Gln Trp Leu Gln Ala

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Asp Val Glu Asp Ala Ala Met Asn Tyr Arg Gly Phe Thr Phe Pro Leu

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Ser His Gln Gln Gln Leu Arg Met Phe Asn Gln Leu Asp Ser His Asp

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Tyr Gly Asp Glu Val Gly Leu Asp Gly Lys Asn Asp Pro Phe Cys Arg

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Lys Pro Phe Pro Trp Gln Val Glu Lys Gln Asp Thr Ala Leu Phe Ala

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His Gly Gly Cys Gln Val Leu Tyr Ala Glu Asp Asn Val Val Val Phe

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Glu Ala Cys Glu Val Val Leu Pro Ala Ser Pro Phe Leu Asn Ala Val

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Gln Trp Gln Cys Lys Glu Gly His Gly Gln Leu Thr Asp Gly Ile Leu

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Ala Leu Pro Ala Ile Ser Ala Thr Val Trp Met Asn

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Met Thr Thr Gln Thr Thr Pro Ala His Ile Ala Met Phe Ser Ile Ala

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Ala Arg Gly His Arg Val Thr Tyr Ala Ile Pro Pro Val Phe Ala Asp

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Ala Asp Ala Tyr Ala Asp Asp Ile Pro Asp Leu Val Leu His Asp Ile

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Thr Ser Tyr Pro Ala Arg Val Leu Ala Arg Arg Trp Gly Val Pro Ala

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Val Ser Leu Phe Pro Asn Leu Val Ala Trp Lys Gly Tyr Glu Glu Glu

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Val Ala Glu Pro Met Trp Arg Glu Pro Arg Gln Thr Glu Arg Gly Arg

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Ala Tyr Tyr Ala Arg Phe Glu Ala Trp Leu Lys Glu Asn Gly Ile Thr

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Glu His Pro Asp Thr Phe Ala Ser His Pro Pro Arg Ser Leu Val Leu

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Ile Pro Lys Ala Leu Gln Pro His Ala Asp Arg Val Asp Glu Asp Val

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Tyr Thr Phe Val Gly Ala Cys Gln Gly Asp Arg Ala Glu Glu Gly Gly

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Trp Gln Arg Pro Ala Gly Ala Glu Lys Val Val Leu Val Ser Leu Gly

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Ser Val Phe Thr Lys Gln Pro Ala Phe Tyr Arg Glu Cys Val Arg Ala

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Phe Gly Asn Leu Pro Gly Trp His Leu Val Leu Gln Ile Gly Arg Lys

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Val Thr Pro Ala Glu Leu Gly Glu Leu Pro Asp Asn Val Glu Val His

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Asp Trp Val Pro Gln Leu Ala Ile Leu Arg Gln Ala Asp Leu Phe Val

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Thr His Ala Gly Ala Gly Gly Ser Gln Glu Gly Leu Ala Thr Ala Thr

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Pro Met Ile Ala Val Pro Gln Ala Val Asp Gln Phe Gly Asn Ala Asp

325 330 335

Met Leu Gln Gly Leu Gly Val Ala Arg Lys Leu Ala Thr Glu Glu Ala

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Thr Ala Asp Leu Leu Arg Glu Thr Ala Leu Ala Leu Val Asp Asp Pro

355 360 365

Glu Val Ala Arg Arg Leu Arg Arg Ile Gln Ala Glu Met Ala Gln Glu

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Gly Gly Thr Arg Arg Ala Ala Asp Leu Ile Glu Ala Glu Leu Pro Ala

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Arg His Glu Arg Gln Glu Pro Val Gly Asp Arg Pro Asn Gly Gly

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Met Thr Thr Gln Thr Thr Pro Ala His Ile Ala Met Phe Ser Ile Ala

1 5 10 15

Ala His Gly His Val Asn Pro Ser Leu Glu Val Ile Arg Glu Leu Val

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Ala Arg Gly His Arg Val Thr Tyr Ala Ile Pro Pro Val Phe Ala Asp

35 40 45

Lys Val Ala Ala Thr Gly Ala Arg Pro Val Leu Tyr His Ser Thr Leu

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Pro Gly Pro Asp Ala Asp Pro Glu Ala Trp Gly Ser Thr Leu Leu Asp

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Asn Val Glu Pro Phe Leu Asn Asp Ala Ile Gln Ala Leu Pro Gln Leu

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Ala Asp Ala Tyr Ala Asp Asp Ile Pro Asp Leu Val Leu His Asp Ile

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Thr Ser Tyr Pro Ala Arg Val Leu Ala Arg Arg Trp Gly Val Pro Ala

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Val Ser Leu Ser Pro Asn Leu Val Ala Trp Lys Gly Tyr Glu Glu Glu

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Val Ala Glu Pro Met Trp Arg Glu Pro Arg Gln Thr Glu Arg Gly Arg

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Ala Tyr Tyr Ala Arg Phe Glu Ala Trp Leu Lys Glu Asn Gly Ile Thr

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Glu His Pro Asp Thr Phe Ala Ser His Pro Pro Arg Ser Leu Val Leu

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Ile Pro Lys Ala Leu Gln Pro His Ala Asp Arg Val Asp Glu Asp Val

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Tyr Thr Phe Val Gly Ala Cys Gln Gly Asp Arg Ala Glu Glu Gly Gly

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Ser Ala Phe Thr Lys Gln Pro Ala Phe Tyr Arg Glu Cys Val Arg Ala

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Phe Gly Asn Leu Pro Gly Trp His Leu Val Leu Gln Ile Gly Arg Lys

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Val Thr Pro Ala Glu Leu Gly Glu Leu Pro Asp Asn Val Glu Val His

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

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Thr His Ala Gly Met Gly Ser Thr Met Glu Ala Leu Ser Asn Ala Val

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Pro Met Ile Ala Val Pro Gln Ala Val Asp Gln Phe Gly Asn Ala Asp

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Met Leu Gln Gly Leu Gly Val Ala Arg Lys Leu Ala Thr Glu Glu Ala

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Thr Ala Asp Leu Leu Arg Glu Thr Ala Leu Ala Leu Val Asp Asp Pro

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Glu Val Ala Arg Arg Leu Arg Arg Ile Gln Ala Glu Met Ala Gln Glu

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Gly Gly Thr Arg Arg Ala Ala Asp Leu Ile Glu Ala Glu Leu Pro Ala

385 390 395 400

Arg His Glu Arg Gln Glu Pro Val Gly Asp Arg Pro Asn Gly Gly

405 410 415

<210> 4

<211> 408

<212> PRT

<213> GT-5 amino acid sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

Met Thr Ser Glu His Arg Ser Ala Ser Val Thr Pro Ala His Ile Ala

1 5 10 15

Met Phe Ser Ile Ala Ala His Gly His Val Asn Pro Ser Leu Glu Val

20 25 30

Ile Arg Glu Leu Val Ala Arg Gly His Arg Val Thr Tyr Ala Ile Pro

35 40 45

Pro Val Phe Ala Asp Lys Val Ala Ala Thr Gly Ala Arg Pro Val Leu

50 55 60

Tyr His Ser Thr Leu Pro Lys Pro Ser Asn Pro Glu Glu Ser Trp Pro

65 70 75 80

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

85 90 95

Ala Leu Pro Gln Leu Ala Asp Ala Tyr Ala Asp Asp Ile Pro Asp Leu

100 105 110

Val Leu His Asp Ile Thr Ser Tyr Pro Ala Arg Val Leu Ala Arg Arg

115 120 125

Trp Gly Val Pro Ala Val Ser Leu Ser Pro Asn Leu Val Ala Trp Lys

130 135 140

Gly Tyr Glu Glu Glu Val Ala Glu Pro Met Trp Arg Glu Pro Arg Gln

145 150 155 160

Thr Glu Arg Gly Arg Ala Tyr Tyr Ala Arg Phe Glu Ala Trp Leu Lys

165 170 175

Glu Asn Gly Ile Thr Glu His Pro Asp Thr Phe Ala Ser His Pro Pro

180 185 190

Arg Ser Leu Val Leu Ile Pro Lys Ala Leu Gln Pro His Ala Asp Arg

195 200 205

Val Asp Glu Asp Val Tyr Thr Phe Val Gly Ala Cys Gln Gly Asp Arg

210 215 220

Ala Glu Glu Gly Gly Trp Gln Arg Pro Ala Gly Ala Glu Lys Val Val

225 230 235 240

Leu Val Ser Leu Gly Ser Ala Phe Thr Lys Gln Pro Ala Phe Tyr Arg

245 250 255

Glu Cys Val Arg Ala Phe Gly Asn Leu Pro Gly Trp His Leu Val Leu

260 265 270

Gln Ile Gly Arg Lys Val Thr Pro Ala Glu Leu Gly Glu Leu Pro Pro

275 280 285

Asn Val Glu Val His Gln Trp Val Pro Gln Leu Asp Ile Leu Thr Lys

290 295 300

Ala Ser Ala Phe Ile Thr His Ala Gly Met Gly Ser Thr Met Glu Ala

305 310 315 320

Leu Ser Asn Ala Val Pro Met Ile Ala Val Pro Gln Ala Val Asp Gln

325 330 335

Phe Gly Asn Ala Asp Met Leu Gln Gly Leu Gly Val Ala Arg Lys Leu

340 345 350

Ala Thr Glu Glu Ala Thr Ala Asp Leu Leu Arg Glu Thr Ala Leu Ala

355 360 365

Leu Val Asp Asp Pro Glu Val Ala Arg Arg Leu Arg Arg Ile Gln Ala

370 375 380

Glu Met Ala Gln Glu Gly Gly Thr Arg Arg Ala Ala Asp Leu Ile Glu

385 390 395 400

Ala Glu Leu Pro Ala Arg His Gly

405

<210> 5

<211> 808

<212> PRT

<213> amino acid sequence of sucrose synthase AtSUS1 (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu

1 5 10 15

Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu

20 25 30

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

35 40 45

Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu

50 55 60

Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile

65 70 75 80

Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu

100 105 110

Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val

115 120 125

Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser

165 170 175

Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys

180 185 190

Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His

195 200 205

Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr

210 215 220

Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg

225 230 235 240

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

245 250 255

Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu

260 265 270

Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

305 310 315 320

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

325 330 335

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

340 345 350

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

370 375 380

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

385 390 395 400

Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

530 535 540

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn

545 550 555 560

Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe

565 570 575

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

580 585 590

Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val

595 600 605

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

610 615 620

Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly

625 630 635 640

Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu

645 650 655

Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala

660 665 670

Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly

675 680 685

Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val

690 695 700

His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala

705 710 715 720

Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser

725 730 735

His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys

740 745 750

Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val

755 760 765

Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg

770 775 780

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln

785 790 795 800

Ala Val Pro Leu Ala Gln Asp Asp

805

<210> 6

<211> 1815

<212> DNA

<213> nucleotide sequence of MalZ (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

atgttaaatg catggcacct gccggtgccc ccatttgtta aacaaagcaa agatcaactg 60

ctcataacac tgtggctgac gggcgaagac ccaccgcagc gcattatgct gcgtacagaa 120

cacgataacg aagaaatgtc agtaccaatg cataagcagc gcagtcagcc gcagccaggc 180

gtcaccgcat ggcgtgcggc gattgatctc tccagcggac aaccccggcg gcgttacagt 240

ttcaaactgc tgtggcacga tcgccagcgt tggtttacac cgcagggctt cagccgaatg 300

ccgccggcac gactggagca gtttgccgtc gatgtaccgg atatcggccc acaatgggct 360

gcggatcaga ttttttatca gatcttccct gatcgttttg cgcgtagtct tcctcgtgaa 420

gctgaacagg atcatgtcta ttaccatcat gcagccggac aagagatcat cttgcgtgac 480

tgggatgaac cggtcacggc gcaggcgggc ggatcaacgt tctatggcgg cgatctggac 540

gggataagcg aaaaactgcc gtatctgaaa aagcttggcg tgacagcgct gtatctcaat 600

ccggtgttta aagctcccag cgtacataaa tacgataccg aggattatcg ccatgtcgat 660

ccgcagtttg gcggtgatgg ggcgttgctg cgtttgcgac acaatacgca gcagctggga 720

atgcggctgg tgctggacgg cgtgtttaac cacagtggcg attcccatgc ctggtttgac 780

aggcacaatc gtggcacggg gggagcttgt cacaaccccg aatcgccctg gcgcgactgg 840

tactcgttta gtgatgatgg cacggcgctc gactggcttg gctatgccag cttgccgaag 900

ctggattatc agtcggaaag tctggtgaat gaaatttatc gcggggaaga cagtattgtc 960

cgccattggc tgaaagcgcc gtggagtatg gacggctggc ggctggatgt ggtgcatatg 1020

ctgggggagg cgggtggggc gcgcaataat atgcagcacg ttgctgggat caccgaagcg 1080

gcgaaagaaa cccagccgga agcgtatatt gtcggcgaac attttggcga tgcacggcaa 1140

tggttacagg ccgatgtgga agatgccgcc atgaactatc gtggcttcac attcccgttg 1200

tggggatttc ttgccaatac cgatatctct tacgatccgc agcaaattga tgcccaaacc 1260

tgtatggcct ggatggataa ttaccgcgca gggctttctc atcaacaaca attacgtatg 1320

tttaatcagc tcgacagcca cgatactgcg cgatttaaaa cgctgctcgg tcgggatatt 1380

gcgcgcctgc cgctggcggt ggtctggctg ttcacctggc ctggtgtacc gtgcatttat 1440

tacggtgatg aagtaggact ggatggcaaa aacgatccgt tttgccgtaa accgttcccc 1500

tggcaggtgg aaaagcagga tacggcgtta ttcgcgctgt accagcgaat gattgcgctg 1560

cgtaagaaaa gtcaggcgct acgtcatggc ggttgtcagg tgctgtatgc ggaagataac 1620

gtggtggtat ttgtccgcgt gctgaatcag caacgtgtac tggtggcaat caaccgtggc 1680

gaggcctgtg aagtggtgct acccgcgtca ccgtttctca atgccgtgca atggcaatgc 1740

aaagaagggc atgggcaact gactgacggg attctggctt tgcctgccat ttcggctacg 1800

gtatggatga actaa 1815

<210> 7

<211> 1248

<212> DNA

<213> nucleotide sequence of Oled-ASP (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 7

atgaccaccc agaccactcc cgcccacatc gccatgttct ccatcgccgc ccacggccat 60

gtgaacccca gcctggaggt gatccgtgaa ctcgtcgccc gcggccaccg ggtcacgtac 120

gccattccgc ccgtcttcgc cgacaaggtg gccgccaccg gcgcccggcc cgtcctctac 180

cactccaccc tgcccggcac cgacgccgac ccggaggcat ggggaagcac cctgctggac 240

aacgtcgaac cgttcctgaa cgacgcgatc caggcgctcc cgcagctcgc cgatgcctac 300

gccgacgaca tccccgatct cgtcctgcac gacatcacct cctacccggc ccgcgtcctg 360

gcccgccgct ggggcgtccc ggcggtctcc ctctttccga acctcgtcgc ctggaagggt 420

tacgaggagg aggtcgccga gccgatgtgg cgcgaacccc ggcagaccga gcgcggacgg 480

gcctactacg cccggttcga ggcatggctg aaggagaacg ggatcaccga gcacccggac 540

acgttcgcca gtcatccgcc gcgctccctg gtgctcatcc cgaaggcgct ccagccgcac 600

gccgaccggg tggacgaaga cgtgtacacc ttcgtcggcg cctgccaggg agaccgcgcc 660

gaggaaggcg gctggcagcg gcccgccggc gcggagaagg tcgtcctggt gtcgctcggc 720

tcggttttca ccaagcagcc cgccttctac cgggagtgcg tgcgcgcctt cgggaacctg 780

cccggctggc acctcgtcct ccagatcggc cggaaggtga cccccgccga actgggggag 840

ctgccggaca acgtggaggt gcacgactgg gtgccgcagc tcgcgatcct gcgccaggcc 900

gatctgttcg tcacccacgc gggcgccggc ggcagccagg aggggctggc caccgcgacg 960

cccatgatcg ccgtaccgca ggccgtcgac cagttcggca acgccgacat gctccaaggg 1020

ctcggcgtcg cccggaagct ggcgaccgag gaggccaccg ccgacctgct ccgcgagacc 1080

gccctcgctc tggtggacga cccggaggtc gcgcgccggc tccggcggat ccaggcggag 1140

atggcccagg agggcggcac ccggcgggcg gccgacctca tcgaggccga actgcccgcg 1200

cgccacgagc ggcaggagcc ggtgggcgac cgacccaacg gtgggtga 1248

<210> 8

<211> 1248

<212> DNA

<213> GT-2 nucleotide sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

atgaccaccc agaccacgcc ggcccatatc gcgatgttca gcatcgccgc ccatggccat 60

gtgaatccga gtctggaagt gatccgtgaa ctggttgccc gtggccatcg cgtgacctat 120

gcgatcccgc cggtgttcgc cgataaagtt gccgccaccg gtgcccgtcc ggttctgtac 180

cacagtacgc tgccgggtcc agatgccgac ccagaagcgt ggggcagtac gctgctggat 240

aacgttgaac cgttcctcaa cgacgcgatc caagcgctgc cacagctggc ggatgcgtat 300

gcggatgaca tcccagatct ggttctccac gatatcacca gctatccagc gcgtgttctg 360

gcgcgtcgct ggggtgttcc agccgttagt ctgagtccga acctcgttgc gtggaaaggc 420

tatgaggaag aagtggcgga accgatgtgg cgcgaaccgc gtcagacgga acgtggtcgc 480

gcctattatg cgcgctttga ggcgtggctg aaagagaatg gcatcacgga acacccggat 540

acctttgcca gccatccacc acgcagtctg gttctgatcc caaaagcgct gcaaccgcat 600

gccgatcgcg tggatgagga cgtgtacacc tttgtgggtg cgtgccaagg tgaccgtgcg 660

gaagaaggtg gttggcaacg tccggcgggt gccgaaaagg ttgttctggt tagtctgggc 720

agcgccttca ccaaacagcc agccttttac cgcgaatgcg tgcgcgcctt cggtaatctg 780

ccgggctggc atctggttct gcaaatcggc cgcaaagtga ccccagcgga actgggtgaa 840

ctgccagata acgtggaggt gcatgactgg gttccgcagc tggatattct gaccaaagcg 900

agcgcgttca tcacgcatgc cggtatgggc agcaccatgg aagcgctgag caatgccgtt 960

ccgatgatcg cggttccgca agccgtggat caattcggca atgcggatat gctgcaaggt 1020

ctgggtgttg cgcgcaaact ggcgacggaa gaggccacgg ccgatctgct gcgtgaaacc 1080

gcgctggcgc tggtggatga tccggaagtt gcccgccgtc tgcgtcgtat tcaagccgag 1140

atggcgcaag aaggtggtac ccgtcgcgcc gccgatctga ttgaagccga actgccagcg 1200

cgccatgaac gccaagaacc agttggtgac cgcccgaatg gcggttaa 1248

<210> 9

<211> 1227

<212> DNA

<213> nucleotide sequence of GT-5 (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

atgaccagtg agcatcgtag tgccagcgtt accccggcac atatcgccat gtttagcatc 60

gccgcacacg gtcacgtgaa tccgagcctg gaagttattc gcgaactggt ggcacgtggc 120

caccgtgtta cctacgccat tccgcctgtt ttcgccgata aagttgccgc aaccggtgca 180

cgtccggtgc tgtaccatag caccctgccg aaaccgagta atccggaaga aagctggccg 240

gaagatcagg aaagcgccat gggcctgttt ctgaatgacg ccattcaggc actgccgcag 300

ttagccgatg cctacgccga tgatatccct gatctggtgc tgcacgatat caccagttat 360

ccggcacgtg ttctggcacg tcgctggggt gtgcctgccg tgagcctgag cccgaatctg 420

gtggcctgga aaggctacga agaagaagtt gccgagccga tgtggcgtga accgcgtcag 480

acagaacgtg gtcgcgccta ctatgcccgc ttcgaagcct ggctgaaaga gaacggcatc 540

accgaacatc cggatacctt cgcaagccat ccgccgcgca gtctggttct gatcccgaaa 600

gccctgcagc cgcatgccga tcgtgtggat gaggacgttt acaccttcgt tggcgcctgt 660

cagggtgatc gtgccgaaga aggtggctgg cagcgccctg caggtgcaga gaaagtggtg 720

ctggtgagcc tgggcagtgc ctttaccaag cagccggcat tctatcgcga atgtgtgcgt 780

gcctttggca acctgccggg ctggcacctg gttctgcaga tcggccgtaa agtgaccccg 840

gccgaactgg gtgaactgcc gcctaatgtg gaagtgcatc agtgggttcc gcagctggat 900

atcctgacca aagccagtgc cttcatcacc catgcaggta tgggcagcac aatggaagcc 960

ctgagtaatg ccgttccgat gatcgccgtt ccgcaggccg tggaccagtt tggcaacgca 1020

gatatgctgc agggtctggg cgtggcacgt aaactggcca ccgaagaagc aaccgcagat 1080

ctgctgcgtg agaccgccct ggccctggtt gacgatccgg aagttgcccg tcgcctgcgt 1140

cgtattcagg ccgaaatggc acaggaaggt ggcacccgtc gtgcagccga tctgattgag 1200

gccgaactgc cggcccgtca tggctaa 1227

<210> 10

<211> 2445

<212> DNA

<213> nucleotide sequence of sucrose synthase AtSUS1 (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 10

atggccaatg cagagcgcat gatcacacgt gtgcacagtc aacgtgaacg tctgaacgag 60

accctggtta gcgagcgcaa cgaggttctg gcactgttaa gccgcgtgga agcaaagggc 120

aaaggcatcc tgcagcaaaa ccagatcatt gccgagtttg aagccctgcc ggaacagacc 180

cgtaagaagc tggagggtgg cccgttcttt gatctgctga aaagcaccca ggaagcaatt 240

gtgttacctc cgtgggtggc actggcagtt cgtccgcgtc cgggcgtttg ggaatacctg 300

cgtgtgaatc tgcatgcact ggtggttgag gagctgcagc ctgccgaatt cctgcatttc 360

aaggaagaac tggtggatgg cgttaaaaac ggtaatttta cattagagct ggactttgaa 420

ccgtttaatg ccagcattcc gcgcccgacc ctgcataaat atatcggtaa cggcgtggat 480

tttctgaatc gccatctgag cgccaagctg tttcatgaca aggagagctt actgcctctg 540

ctgaaatttc tgcgtctgca tagtcaccag ggcaagaacc tgatgctgag cgaaaagatc 600

caaaatctga acaccctgca gcacaccctg cgtaaagccg aggaatatct ggccgaactg 660

aagagcgaaa ccctgtatga ggagtttgag gccaagttcg aggagatcgg cctggagcgt 720

ggctggggtg acaacgcaga acgtgtgctg gacatgattc gtctgctgct ggacctgctg 780

gaggcaccgg atccgtgcac actggagaca ttcctgggcc gcgtgccgat ggttttcaat 840

gtggtgattc tgagcccgca cggctacttt gcacaggaca acgttctggg ttatccggat 900

acaggtggcc aagtggtgta cattctggat caggtgcgtg ccttagagat cgagatgctg 960

cagcgcatta aacagcaggg cctgaatatc aaaccgcgca tcctgatcct gacccgtctg 1020

ttacctgatg ccgtgggcac aacctgcggt gaacgcctgg aacgcgtgta tgatagcgaa 1080

tactgtgaca ttctgcgtgt gccgttccgt acagagaaag gcatcgtgcg taaatggatt 1140

agccgcttcg aggtttggcc ttacctggaa acctacaccg aggatgcagc agtggagtta 1200

agcaaagagc tgaacggcaa gccggacctg attattggca actacagcga cggcaacctg 1260

gtggccagcc tgttagccca caaattaggt gtgacccagt gtaccatcgc ccacgcactg 1320

gaaaagacaa aatacccgga cagcgatatc tactggaaaa agttagatga taaatatcac 1380

ttcagctgcc agtttaccgc cgacatcttt gccatgaacc acaccgattt tattatcaca 1440

agcacattcc aggaaatcgc aggcagtaaa gagaccgttg gccagtacga gagccataca 1500

gcctttacac tgcctggcct gtatcgtgtt gtgcacggca tcgatgtgtt tgatcctaaa 1560

tttaacattg ttagcccggg tgcagacatg agtatctact tcccgtacac cgaggagaag 1620

cgccgtctga ccaagtttca cagtgaaatc gaggaactgc tgtacagtga cgtggagaac 1680

aaggagcatc tgtgcgtgtt aaaggataag aaaaaaccga tcttatttac aatggcacgc 1740

ctggatcgcg tgaagaatct gagtggcctg gttgagtggt atggcaaaaa tacccgcctg 1800

cgcgaactgg ccaatctggt tgtggttggt ggcgaccgtc gtaaagaaag caaggacaac 1860

gaggagaagg ccgagatgaa gaaaatgtac gatctgatcg aagagtataa gctgaatggc 1920

cagtttcgct ggatcagcag tcagatggac cgtgtgcgca atggcgaact gtatcgctac 1980

atttgtgaca caaagggcgc attcgttcag ccggcactgt atgaggcctt cggcctgaca 2040

gtggtggaag ccatgacctg cggcctgccg acctttgcaa cctgcaaagg cggcccggca 2100

gaaatcatcg ttcatggcaa gagcggcttc catatcgatc cgtatcatgg tgaccaggcc 2160

gccgatacac tggcagactt ttttaccaaa tgtaaagagg atccgagcca ctgggatgag 2220

attagcaagg gtggtctgca gcgcatcgaa gaaaaataca cctggcagat ctacagccaa 2280

cgtctgctga ccctgaccgg tgtgtatggt ttctggaaac acgtgagtaa tctggaccgt 2340

ctggaagccc gccgttacct ggaaatgttc tatgcactga aatatcgccc gctggcacaa 2400

gccgttcctc tggcacaaga cgatcatcac catcaccatc attaa 2445

<210> 11

<211> 70

<212> DNA

<213> malZ-homo-F(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

tgcattaggc tatggcaagg tgatcagatt ttcatcacag gggaattatg gtgtaggctg 60

gagctgcttc 70

<210> 12

<211> 70

<212> DNA

<213> malZ-homo-R(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

gttttatccg cggatgatgg cgcaggcgtc acgcaaggcg ttataaaacg atgggaatta 60

gccatggtcc 70

Claims

1. A method for determining glycosyltransferase activity, characterized in that fructose generated in a double-enzyme reaction system of UDP-glucose dependent glycosyltransferase coupled sucrose synthase is determined based on a chromogenic method to characterize the glycosyltransferase activity; the UDP-glucose dependent glycosyltransferase and sucrose synthase can be expressed in E.coli alone or in combination; the escherichia coli is an engineering strain E.coli malZ-KO obtained by knocking out a maltodextrin glucosidase coding gene malZ from E.coli BL21 (DE 3); wherein the sucrose synthase is SUS1, and the amino acid sequence is SEQ ID NO. 5.

2. The method for measuring glycosyltransferase activity according to claim 1, wherein the UDP-glucose dependent glycosyltransferase and sucrose synthase are pure enzymes or crude enzyme extracts.

3. The method for measuring glycosyltransferase activity according to claim 1, wherein the color reaction is a DNS reaction.

4. Use of a glycosyltransferase activity assay according to claim 1 in an assay for the activity of UDP-glucose dependent glycosyltransferases, in a screening of novel UDP-glucose dependent glycosyltransferases or in an activity screening of UDP-glucose dependent glycosyltransferase mutations.

5. The use according to claim 4, wherein the UDP-glucose dependent glycosyltransferase is Oled-ASP having the amino acid sequence of SEQ ID NO. 2.