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A kind of tea tree sucrose synthase cssus587, preparation method and application

A technology of sucrose synthase and tea tree, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of high price of UDP-Glucose and reduced reaction rate, so as to improve synthesis efficiency and reduce production cost , the effect of high purity

Active Publication Date: 2022-07-19
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The corresponding glycoside and UDP are generated through the catalyzed reaction of GT enzyme, but UDP-Glucose is expensive, and UDP, as one of the products of the reaction of glycoside preparation, will greatly inhibit the activity of glycosyltransferase
As UDP accumulates in the reaction mixture, the reaction rate will gradually decrease

Method used

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  • A kind of tea tree sucrose synthase cssus587, preparation method and application
  • A kind of tea tree sucrose synthase cssus587, preparation method and application
  • A kind of tea tree sucrose synthase cssus587, preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A preparation method of tea tree sucrose synthase CsSUS587, comprising the following steps:

[0036] Target gene cloning: Using tea tree cDNA as a template, specific primers were designed with SnapGene Viewer software, and then PCR reaction was performed; PCR products were detected by 1.2% agarose gel electrophoresis, and a band with a size of about 2000bp was obtained, that is, the cloning was successful.

[0037] Among them, the primer sequence:

[0038] SalI: 5'-gatatcggatccgaattcgagctccgtcgacgcatggcatctcatgttctga-3', as shown in SEQID NO.1;

[0039] Notl: 5'-gatctcagtggtggtggtggtggtgctcgagtgcggccgcctactcaatagccaaagggacttctg-3', as shown in SEQ ID NO.2;

[0040] PCR reaction system: 2 μL of cDNA template, 2 μL of 10 μM upstream and downstream primers, 24 μL of high-fidelity polymerase (10×Taq mix enzyme), ddH 2 Add O to 50 μL;

[0041]PCR reaction conditions: 30 cycles of pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30s, annealing at 55°C for 30s, ...

Embodiment 2

[0067] In order to verify the application effect of the present application, 3-site kaempferol glycosides were synthesized using the application method of Example 1. The only difference in synthesis in 1 is that no tea tree sucrose synthase (CsSUS587) was added, and the 3-site kaempferol glycosides obtained by the method in Example 1 were detected by high performance liquid chromatography, and the kaempferol glycosides were extracted under positive ion mode. Molecular weight 449. The result is as Figure 1-2 : The production of kaempferol glycosides at 3 sites was significantly increased after the addition of tea tree sucrose synthase CsSUS587.

Embodiment 3

[0069] In order to verify the application effect of the application, the application method of Example 1 is utilized to synthesize phenethyl glucoside, respectively to the existing utilization of GT to synthesize phenethyl glucoside (the synthetic difference with the embodiment 1 is only that the tea tree sucrose synthase CsSUS587 is not added) , The phenethyl glucoside obtained by the method in Example 1 was detected by high performance liquid chromatography, and the molecular weight of the phenethyl glucoside synthesized in the positive ion mode was 307. The result is as Figure 3-5 : image 3 The results showed that the amount of phenylethanol glycosides was significantly increased after the addition of tea tree sucrose synthase CsSUS587. Figure 4 for right image 3 Perform mass spectrometry analysis. Figure 5 for right image 3 The peak area comparison results were performed after two technical replicates. In addition, the effect on n-octanol glycosides is also good...

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Abstract

The invention belongs to the technical field of molecular biology, and in particular relates to a tea tree sucrose synthase CsSUS587, a preparation method and an application, comprising using tea tree cDNA as a template, designing primers, and PCR amplifying a target gene; Transform into competent cells; separation and purification of tea tree sucrose synthase. The sucrose synthase CsSUS587 prepared by the invention has high purity, can promote uridine diphosphate and sucrose to catalyze the generation of uridine diphosphate glucose which can be used for glycoside synthesis, and can be used in combination with glycosyltransferase to improve the synthesis efficiency of corresponding glycosides.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and particularly relates to a tea tree sucrose synthase CsSUS587, a preparation method and an application. Background technique [0002] Sucrose synthase (SUS) is an important cytoplasmic enzyme in plant cells, which plays a key role in the process of sugar metabolism in plants. SUS is a reversible reaction enzyme that can catalyze the decomposition of sucrose, and UDP-Glucose and fructose are its catalytic products. The study of the SUS gene has been carried out extensively in some plants, especially in cotton. Studies have shown that by inhibiting the expression of SUS, the initiation and elongation of cotton fiber cells are significantly affected. The reason may be that glucose is a reactant for cellulose synthase for the production of cellulose for cell wall biosynthesis, a phenomenon that suggests that SUS is an essential key factor in cellulose synthesis and cell wall formation....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/30
Inventor 宋传奎许淼晶荆婷婷张娜靳洁阳赵明月
Owner ANHUI AGRICULTURAL UNIVERSITY
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