阅读说明：本技术 一种糖基转移酶活性测定新方法 (Novel method for determining glycosyl transferase activity ) 是由 吴旭日 李萌 夏媛 杜雅丽 赵玲 陈依军 于 2021-01-04 设计创作，主要内容包括：本发明属于生物催化技术领域,具体涉及一种糖基转移酶活性测定新方法,其特征在于通过显色法测定UDP-葡萄糖依赖糖基转移酶偶联蔗糖合酶的双酶反应体系中生成的果糖,以表征糖基转移酶的活性。本发明公开了一种新的基于颜色反应的UDP-葡萄糖依赖糖基转移酶的筛选方法,为糖基转移酶的改造和发掘提供了一种初步活性筛选工具,也为UDP-葡萄糖依赖糖基转移酶的活性表征提供了新的方法。(The invention belongs to the technical field of biocatalysis, and particularly relates to a novel method for measuring glycosyl transferase activity, which is characterized in that fructose generated in a two-enzyme reaction system of UDP-glucose-dependent glycosyl transferase coupled sucrose synthase is measured by a color development method to characterize the activity of glycosyl transferase. The invention discloses a novel screening method of UDP-glucose-dependent glycosyltransferase based on color reaction, provides a primary activity screening tool for the transformation and the discovery of glycosyltransferase, and also provides a novel method for the activity characterization of UDP-glucose-dependent glycosyltransferase.)

1. A novel method for measuring the activity of glycosyltransferase is characterized in that the fructose generated in a two-enzyme reaction system of UDP-glucose-dependent glycosyltransferase coupled with sucrose synthase is measured based on a color development method to characterize the activity of glycosyltransferase.

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

3. The novel method for assaying glycosyltransferase activity according to claim 2, characterized in that the UDP-glucose dependent glycosyltransferase and sucrose synthase can be expressed separately or co-expressed in e.coli, streptomyces, yeast, bacillus subtilis, bacteriophage and CHO cell expression systems.

4. The novel method for assaying glycosyltransferase activity of claim 3, wherein the E.coli is E.coli malZ-KO, an engineered strain of E.coli BL21(DE3) knock-out maltodextrine glucosidase encoding gene malZ.

5. The novel method for assaying glycosyltransferase activity of claim 4, wherein the color reaction is a DNS reaction, a fiyline reagent reaction, a benedict reagent reaction, or a barfoed reagent reaction.

6. The novel method for glycosyltransferase activity of claim 5, wherein the method is used for the determination of UDP-glucose-dependent glycosyltransferase activity, screening of novel UDP-glucose-dependent glycosyltransferases, screening of UDP-glucose-dependent glycosyltransferase mutated activity.

7. The novel method of measuring glycosyltransferase activity of claim 6, wherein the UDP-glucose-dependent glycosyltransferase is OleD-ASP having the amino acid sequence of SEQ ID NO. 2; the sucrose synthase is SUS1, and its amino acid sequence is SEQ ID NO: 5.

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 sugar molecules to be transferred to different acceptor substrates, and is widely applied to the fields of disease treatment target research, drug development and the like. UDP-glucose dependent glycosyltransferases (UGTs) use uridine diphosphate glucose (UDPG) as glycosyl donors, can selectively catalyze natural products and non-natural compounds to synthesize glycosylation products with different biological activities and physicochemical properties, and are one of important tool enzymes for drug and food additive development. With the continuous deepening of UGTs research and the continuous improvement of the excavation difficulty of new enzymes, the application of wild type UGTs with weak catalytic activity and narrow substrate spectrum in glycosylation modification of natural products and non-natural compounds is limited (Li C, et al. trends Biotechnol.2020,38: 729-. At present, random mutation and directed evolution are important strategies for improving UGTs catalytic properties and designing artificial new enzymes, however, development of an efficient screening method capable of representing UGTs and mutant activities thereof is one of technical difficulties hindering UGTs modification.

In order to solve the problems, researchers have developed some UGTs screening methods, but all have the disadvantages and are difficult to effectively apply: (1) radiolabelling is highly sensitive, but the procedure is complicated and leads to radioactive chemicals (Brown C, et al. nat Protoc,2012,7: 1634-; (2) immunological methods are also very sensitive, but are limited by the cost of detection and stability of antibody binding to the receptor (Chokhawala HA, et al. ACS Chem Biol,2008,3: 567-; (3) the advantages of chromatography and mass spectrometry in terms of accuracy are obvious, but the cost is high, the time consumption is long, and the high throughput cannot be realized (Kopp M, et al. Chembiolchem, 2007,8: 813-; (4) the screening method based on the pH indicator has poor sensitivity and specificity and multiple interference factors (Deng C, et al. anal Biochem,2004,330: 219-226); (5) the fluorescence-based screening methods require the preparation of complex compounds with complex structures and the fluorescence signals are susceptible to interference (Lee HS, et al. anal Biochem,2011,418: 85-88; Ryu J, et al. Bioorg Med Chem,2014,22: 2571-2575); (6) spectrophotometry combines the UDP or glycosylation product produced by the glycosylation reaction with other enzymatic reactions, is relatively complex and is only suitable for pure enzymes (Li Y, et al, JBiotechnol,201,227: 10-18). Therefore, the UDP-glucose dependent glycosyltransferase activity determination method which is simple, rapid, low in cost and high in throughput is developed, and has important application value for the discovery and modification of glycosyltransferase.

Disclosure of Invention

Object of the Invention

UDP glucose-dependent glycosyltransferase is an important tool enzyme, and shows great application prospect in the aspects of compound structure modification and drug-forming modification. However, mutant screening and new enzyme mining in the UDP glucose-dependent glycosyltransferase modification process involve a large amount of screening work, the existing screening and activity detection methods have certain defects, and in order to overcome the defects, the invention provides a novel method for detecting and screening the glycosyltransferase activity based on color reaction so as to provide a feasible screening tool.

Technical scheme

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

a novel method for measuring the activity of glycosyltransferase is characterized in that the fructose generated in a two-enzyme reaction system of UDP-glucose-dependent glycosyltransferase coupled with sucrose synthase is measured based on a color development method to characterize the activity of glycosyltransferase.

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

The new method for determining the activity of the glycosyltransferase is characterized in that the UDP-glucose-dependent glycosyltransferase and the sucrose synthase can be independently or jointly expressed in an escherichia coli, streptomycete, yeast, bacillus subtilis, phage and CHO cell expression system.

The new method for determining the activity of the glycosyltransferase is characterized in that the escherichia coli is an engineering strain E.coli malZ-KO of E.coli BL21(DE3) knock-out maltodextrin glucosidase encoding gene malZ.

The new method for measuring the activity of the glycosyltransferase is characterized in that the color reaction comprises a DNS reaction, a Fehling reagent reaction, a benedict reagent reaction and a barfoed reagent reaction.

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

The new method for measuring the activity of the glycosyltransferase 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 its amino acid sequence is SEQ ID NO: 5.

In particular to

1) Alpha-glucosidase MalZ for hydrolyzing sucrose to generate fructose in E.coli BL21 is determined by KEGG analysis, and is knocked out to construct an engineering strain E.coli malZ-KO, so that detection background interference is reduced, and a chassis strain is provided for development of the method.

2) Expressing OleD-ASP and sucrose synthase in E.coli malZ-KO respectively or co-expressing, constructing a double-enzyme coupling reaction system, and verifying the feasibility of the UDP-glucose dependent glycosyltransferase activity determination method based on color reaction.

3) Optimizing the catalytic reaction conditions of OleD-ASP and sucrose synthase, a novel method for determining the activity of UDP-glucose-dependent glycosyltransferase based on color reaction is created.

4) The inventor is adopted to apply for a patent in the prior art (application number: 202010921312.1), the amino acid sequences of the UDP-glucosyltransferase GT-2 are SEQ ID NO. 3 and GT-5, the amino acid sequence of the UDP-glucosyltransferase GT-2 is SEQ ID NO. 4, and the coupling sucrose synthase catalyzes the glycosylation modification of nosiheptide to be a model reaction so as to verify the practicability of the method.

Wherein:

1. KEGG (Kyoto Encyclopedia of Genes and genomics, Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database for systematic analysis of gene function, a knowledge base linking genomic information and functional information. One of the cores of the KEGG Pathway is a metabolic Pathway graph drawn by hands according to related knowledge and is divided into 6 types, namely Metabolim, Genetic information Processing, Cellular Processing, Environmental information Processing, organic Systems and Human Diseases. These KEGG pathways contain information on a large number of proteins, compounds, and their interaction relationships.

Starch and sucrose metabolism (00500 Starch and sucrose metabolism) under Carbohydrate metabolism (1.1 Carbohydrate metabolism) were selected in the KEGG metabolic Pathway Database (KEGG Pathway Database).

Through analysis, in a sucrose metabolic pathway of E.coli BL21(DE3), maltodextrin glucosidase (MalZ) can catalyze sucrose to generate fructose and glucose, which may cause background interference on the screening method based on fructose detection in the invention, so that the malZ gene is knocked out.

2. The principle of DNS (dinitrosalicylic acid) color development;

reducing sugar can be oxidized into saccharic acid and other products by heating under alkaline condition, and the oxidant 3, 5-dinitrosalicylic acid is reduced into 3-amino-5-nitro salicylic acid with red brown color. Within a certain range, the amount of reducing sugar is in direct proportion to the shade of the color of the brownish red substance, the optical density value is measured by utilizing a spectrophotometer under the wavelength of 540nm, the standard curve is checked and calculated, and the contents of the reducing sugar and the total sugar in the sample can be obtained.

Advantageous effects

1. The invention discloses a novel UDP-glucose dependent glycosyltransferase activity detection or screening method based on color reaction, 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 a double-enzyme coupling reaction by using a DNS (domain name system) color development method, thereby establishing an activity detection or screening method of glycosyltransferase based on color reaction. In order to achieve the aim, the invention takes UDP-glucose dependent glycosyltransferase OleD-ASP and sucrose synthase coupled catalysis 4-methylumbelliferone glycosylation modification as model reaction, carries out construction of the method, and verifies the applicability of the method by using the reaction of glycosyltransferase catalysis synthesis of glycosylated nosiheptide.

2. The method for rapidly determining the activity of the UDP-glucose-dependent glycosyltransferase based on the color reaction has the advantages of simple and convenient operation, low cost, high flux and the like, can be used for the aspects of determining the activity of the UDP-glucose-dependent glycosyltransferase, screening novel UDP-glucose-dependent glycosyltransferase, screening the mutated activity of the UDP-glucose-dependent glycosyltransferase and the like, and provides a primary screening tool for the discovery and the modification of the glycosyltransferase.

Drawings

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

FIG. 2 is a search of a method for measuring UDP-glucose-dependent glycosyltransferase activity based on a color reaction;

FIG. 3 is a SDS-PAGE analysis of the co-expression of sucrose synthase (A) and OleD-ASP, where M: protein molecular weight standards; lane 1: coli BL21(DE3) control; lane 2: coli malZ-KO control; lanes 3, 4: coli K & 1; lanes 5-6: coli K & 2;

fig. 4 is a comparison of reaction efficiencies of e.coli K &1(a) and e.coli K &2 (B);

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

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

Detailed Description

The following examples illustrate specific steps of the present invention, but the scope of the present invention is not limited by these examples.

Preparing a DNS reagent: dissolving 18.2g of sodium potassium tartrate in 50ml of distilled water, heating, adding 0.63g of 3, 5-dinitrosalicylic acid, 2.1g of NaHO and 0.5g of phenol in the hot solution in sequence, stirring until the mixture is dissolved, cooling, adding distilled water to reach a constant volume of 100ml, storing in a brown bottle, and storing at room temperature.

Example 1 conditions for HPLC analysis

A 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 introduction volume: 2 mu L of the solution; the flow rate of the system: 1.0 mL/min; column temperature: 30 ℃; detection wavelength: 232 nm; elution gradient: linearly eluting 10% B-50% B for 6min, and linearly eluting 50% B-70% B for 16 min.

Example 2 DNS color development 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 and uniformly mixing, and sequentially adding0.3mL of DNS reagent, fully and uniformly mixing, and centrifuging at 12000rpm for 30 s; boiling water bath for 5min, centrifuging at 12000rpm for 30s, cooling to room temperature, and determining OD₅₄₀Value according to fructose concentration and its corresponding OD₅₄₀Values were plotted against a fructose standard curve. The standard curve equation is that y is 0.4497x-0.0345 (r)²0.999) where y is OD_540nmAnd x is the fructose concentration.

TABLE 1 preparation of fructose solution

After the double-enzyme coupling reaction is finished, carrying out 80-degree water bath on the reaction solution for 5min to terminate the reaction, centrifuging, taking 0.3mL of supernate, adding 0.3mL of DNS reagent, fully and uniformly mixing, and centrifuging at 12000rpm for 30 s; boiling water bath for 5min, centrifuging at 12000rpm for 30s, cooling to room temperature, and determining OD_540nmThe value is obtained. When measuring, the measurement solution with too high concentration needs to be diluted to OD_540nmTo within 1.0.

Example 3 Lambda Red Gene recombination method for knocking out malZ Gene

In KEGG metabolic Pathway Database (KEGG Pathway Database), through classification search, selecting Starch and sucrose metabolism (00500 Starch and sucrose metabolism) modules under Carbohydrate metabolism (1.1 Carbohydrate metabolism), selecting strain E.coli BL21 to be analyzed (DE3), obtaining its Starch and sucrose metabolic Pathway map (https:// www.kegg.jp/KEGG-bin/show _ pathwayebl00500), analyzing sucrose catabolic Pathway, determining key enzyme alpha-glucosidase MalZ for sucrose hydrolysis (SEQ ID NO: 1).

The upstream and downstream 50bp of maltodextrin glucosidase gene malZ is selected as a homology arm, and then the homology arm is respectively added to the 5' ends of pKD3 plasmid universal amplification primers P1 and P2, 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’

Using pKD3 plasmid as template, amplifying substitution fragment with homologous arm (the fragment contains FRT site and chloramphenicol resistance gene), separating PCR amplification fragment by 1% agarose gel electrophoresis after PCR reaction, and recovering homologous substitution fragment.

The pKD46 plasmid is transformed to E.coli BL21 competence, the homologous substitution fragment is added to E.coli BL21 electrotransformation competence for homologous recombination, then culture is carried out at 42 ℃ to lose the pKD46 plasmid in the positive recombinant bacteria, then the pCP20 plasmid is transformed to the recombinant bacteria competence, the plasmid eliminates chloramphenicol resistance of bacteria by acting on FRT locus, and culture is carried out at 42 ℃ to lose the plasmid. Coli malZ-KO strain was finally obtained.

Example 4 E.coli malZ-KO background detection

Coli malZ-KO and e.coli BL21 were sonicated and centrifuged to obtain supernatants. According to the total volume of the reaction system of 1mL, the final concentration of each component is respectively as follows: the DNS color reaction was carried out with 0.2mol/L sucrose solution, 0.5mL crude enzyme solution, and 0.05mol/L buffer solution (pH 7.5). The results show that the concentration of fructose produced by E.coli BL21 is 2.326mmol/L, while the concentration of fructose produced by E.coli malZ-KO is 0.074mmol/L, and the fructose concentration is reduced by 31.5 times, so that the background interference of E.coli BL21 is completely eliminated.

Example 5 soluble expression and detection methods of OleD-ASP and sucrose synthase SUS1

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

On the basis of double-enzyme soluble expression, a 5mL reaction system is constructed to verify the feasibility of the UDP-glucose activity determination 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 crude enzyme solution of OleD-ASP, 1.25mL of crude enzyme solution of sucrose synthase SUS1, and 2.5mL of Tris-HCl bufferFlush (pH8.0, containing 5mM MgCl)₂). Reacting for 12 hours at 30 ℃ and 220 r/min. The results are shown in FIG. 2, and the two-enzyme reaction group showed a distinct orange color, OD, compared to the empty control of E.coli malZ-KO_540nmIs 1.37. The results indicate that a UDP-glucose activity assay method for detecting fructose based on the DNS method is feasible.

EXAMPLE 6 construction of Co-expression Strain

Using the co-expression vector pETDuet-1, a co-expression plasmid pETDuet-1-1ATSUS1-oleD-Asp (see FIG. 5) of sucrose synthase (Arabidopsis thaliana sucrose synthase, abbreviated as AtSUS1) and OleD-ASP was constructed with the aid of restriction sites such as Nco I/Hind III and Nde I/Xho I, and E.coli malZ-KO was introduced to obtain a co-expression strain E.coli K&Coli K&2 (table 2). The induced expression condition of the engineering strain IPTG is 0.4mM IPTG, the temperature is 20 ℃, the time is 14 hours, the expression quantity is detected by SDS-PAGE (figure 3), and then a 5mL reaction system is constructed: 4-methylumbelliferone 1mg/ml, UDP-glucose 3.5mg/ml, sucrose 0.2M, crude enzyme solution 2.5ml, 2.5ml Tris-HCl buffer (pH8.0, containing 5mM MgCl 2). And performing DNS color reaction after reacting for 12h at 30 ℃ and 220 r/min. Coli K is shown in FIG. 4&Coli K&2 can normally catalyze sucrose to produce fructose, and E&2 Activity (OD)_540nm1.85) slightly higher than e&1(OD_540nm1.73), so e&2 follow-up studies were carried out.

TABLE 2 Co-expression strains

Example 7 optimization of conditions for the two-enzyme coupling reaction

A two-enzyme reaction system was constructed as in example 6, and the reaction temperature (25 ℃ C. -40 ℃ C.), the reaction pH (6.5-9.0) and the reaction time (2 h-14 h) were respectively examined for OD_540nmAnd the effect of 4-methylumbelliferone conversion. 1) Optimum reaction temperature: OD at 30 ℃_540nmThe maximum value was 1.83. The conversion of 4-methylumbelliferone was 66.5%; 2) optimum reaction pH: pH8.0, OD_540nmThe maximum value was 1.83. Conversion of 4-methylumbelliferoneThe rate was 67.5%; 2) the optimal reaction time is as follows: 10h, OD_540nmThe maximum value was 2.36. The conversion of 4-methylumbelliferone was 82.8%. Therefore, it was finally determined that the optimum conditions for the two-enzyme reaction were 30 ℃ and pH8.0 for 10 hours, as shown in FIG. 6.

EXAMPLE 8 examination of the detection Limit of color reaction

Detection of OD of blank test group (containing no fructose) by DNS color development₅₄₀Value, repeat the assay 10 times. The detection limit was calculated according to the fructose standard curve of example 2, and the calculation formula for the detection limit was as follows:

LOD＝3s/b

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

The detection limit of the color reaction is 0.026mmol/L through calculation.

Example 9 examination of color reaction stability

Treating fructose solution (0.5mmol/L, 0.75mmol/L, 1.0mmol/L, 1.25mmol/L, 1.5mmol/L) with different concentrations, placing in room at normal temperature, and measuring OD for 0min, 10min, 20min, 30min, 45min, and 60min respectively_540nmThe value is obtained. The result shows that the OD of the chromogenic substance 3-amino-5-nitro salicylic acid generated by the action of fructose solutions with different concentrations and DNS reagent is 60min after the chromogenic substance is placed indoors_540nmThe value did not change. This shows that the stability of the color reaction is better.

Example 10 use of the color reaction-based glycosyltransferase screening method

The enzymatic method is used for catalyzing glycosylation modification of nosiheptide as a model reaction so as to verify the practicability of the vitality determination method based on the color reaction. UDP-glucose dependent glycosyltransferases GT-2 and GT-5 of the inventor's earlier invention patent (application No. 202010921312.1) were selected as catalysts, and respectively construct corresponding co-expression plasmids with sucrose synthase AtSUS1, and after E.coli malZ-KO was transformed, co-expression strains E.coli K &3 and E.coli K &4 were obtained, respectively. The conditions for IPTG inducible expression were 0.4mM IPTG, 20 ℃ and 14 hours.

A5 mL reaction was constructed as in example 6: nosiheptide 1mg/ml, UDP-grapeSugar 3.5mg/ml, sucrose 0.2M, crude enzyme solution 2.5ml, Tris-HCl buffer (pH8.0, containing 5mM MgCl)₂). And performing DNS color reaction after reacting for 10h at 30 ℃ and 220 r/min. Coli K&OD of 4 reaction group_540nm1.17, significantly higher than e&0.52 for reaction group 3. The trend of the above results is consistent with the trend of the enzyme activity of the inventor's prior invention patent (application No. 202010921312.1). In conclusion, the UDP-glucose dependent glycosyltransferase activity screening method based on the color reaction can be used for enzyme activity determination, mutant activity screening and the like, and is an effective enzyme activity preliminary screening method.

Sequence listing

<110> university of Chinese pharmacy

<120> novel method for assaying glycosyltransferase activity

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<213> amino acid sequence of MalZ (2 Ambystoma laterale x Ambystoma jeffersonanum)

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Met Leu Asn Ala Trp His Leu Pro Val Pro Pro Phe Val Lys Gln Ser

1 5 10 15

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Pro Met His Lys Gln Arg Ser Gln Pro Gln Pro Gly Val Thr Ala Trp

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Arg Ala Ala Ile Asp Leu Ser Ser Gly Gln Pro Arg Arg Arg Tyr Ser

65 70 75 80

Phe Lys Leu Leu Trp His Asp Arg Gln Arg Trp Phe Thr Pro Gln Gly

85 90 95

Phe Ser Arg Met Pro Pro Ala Arg Leu Glu Gln Phe Ala Val Asp Val

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Pro Asp Ile Gly Pro Gln Trp Ala Ala Asp Gln Ile Phe Tyr Gln Ile

115 120 125

Phe Pro Asp Arg Phe Ala Arg Ser Leu Pro Arg Glu Ala Glu Gln Asp

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

145 150 155 160

Trp Asp Glu Pro Val Thr Ala Gln Ala Gly Gly Ser Thr Phe Tyr Gly

165 170 175

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

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Gly Val Thr Ala Leu Tyr Leu Asn Pro Val Phe Lys Ala Pro Ser Val

195 200 205

His Lys Tyr Asp Thr Glu Asp Tyr Arg His Val Asp Pro Gln Phe Gly

210 215 220

Gly Asp Gly Ala Leu Leu Arg Leu Arg His Asn Thr Gln Gln Leu Gly

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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

275 280 285

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

325 330 335

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

355 360 365

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

370 375 380

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

385 390 395 400

Trp Gly Phe Leu Ala Asn Thr Asp Ile Ser Tyr Asp Pro Gln Gln Ile

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Asp Ala Gln Thr Cys Met Ala Trp Met Asp Asn Tyr Arg Ala Gly Leu

420 425 430

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

435 440 445

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

450 455 460

Leu Ala Val Val Trp Leu Phe Thr Trp Pro Gly Val Pro Cys Ile Tyr

465 470 475 480

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

485 490 495

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

500 505 510

Leu Tyr Gln Arg Met Ile Ala Leu Arg Lys Lys Ser Gln Ala Leu Arg

515 520 525

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

530 535 540

Val Arg Val Leu Asn Gln Gln Arg Val Leu Val Ala Ile Asn Arg Gly

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

565 570 575

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

580 585 590

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

595 600

<210> 2

<211> 415

<212> PRT

<213> amino acid sequence of OleD-ASP (2 Ambystoma laterale x Ambystoma jeffersonanium)

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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 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

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

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Pro Gly Thr 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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

340 345 350

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

370 375 380

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> 3

<211> 415

<212> PRT

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

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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

20 25 30

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

340 345 350

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

370 375 380

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 jeffersonanum)

<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 jeffersonanum)

<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 jeffersonanum)

<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 jeffersonanium)

<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 jeffersonanum)

<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 jeffersonanum)

<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 jeffersonanium)

<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