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Phytoalexin Glycosyltransferase and Its Encoding Gene and Application from Sorbus australis

A technology of glycosyltransferase and phytochemicals, which is applied in the biological field

Active Publication Date: 2020-08-04
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the secondary metabolism of plants, there are many types of glycosyltransferases involved. At present, the most studied glycosyltransferases are plant hormone glycosyltransferases, flavonoid glycosyltransferases and terpene glycosyltransferases. enzymes, etc., but the glycosyltransferases of plant protection have not been reported yet

Method used

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  • Phytoalexin Glycosyltransferase and Its Encoding Gene and Application from Sorbus australis
  • Phytoalexin Glycosyltransferase and Its Encoding Gene and Application from Sorbus australis
  • Phytoalexin Glycosyltransferase and Its Encoding Gene and Application from Sorbus australis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the cloning of Rowan phytoalexin glycosyltransferase gene

[0051] Total RNA was extracted from suspension cells of Sorbus australis, and cDNA was obtained by reverse transcription. Using the obtained cDNA as a template, PCR reaction was carried out using primer 1 and primer 2.

[0052] Primer 1: 5'-GAG GGATCC ATGGGAGACGTAATAGTGCT-3';

[0053] Primer 2: 5'-GAG GCGGCCGC TTATGTAAAGCTATTGACAAAGTTA-3'.

[0054] The PCR reaction system is as follows: KOD-Plus-Neo 2μL; 2mM dNTPs 10μL; 25mM MgSO 4 6 μL; 10×PCR buffer for KOD-Plus-Neo 10 μL; 3 μL each of primer 1 and primer 2; 2 μL of cDNA template; ddH 2 O to make up to 100 μL.

[0055] PCR reaction conditions: 94°C for 10min; 35 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min; 72°C for 10min; 4°C for maintenance.

[0056] After PCR, the obtained PCR products were detected by 1.5% agarose gel electrophoresis and sequenced. Sequencing results showed that the sequence of the PCR product was "5'-GAG ...

Embodiment 2

[0057] Example 2, Exogenous Expression of Rowan Phytoalexin Glycosyltransferase

[0058] 1. Construction of recombinant expression vector

[0059] The PCR product "5'-GAG of embodiment 1 GGATCC +sequence 2+ GCGGCCGC After CTC" is purified and recovered, it is double-digested with restriction endonucleases BamH I and Not I, and the digested product is recovered and connected to the large backbone fragment of the pET-28a(+) plasmid that has undergone the same double-digestion to obtain a recombinant plasmid .

[0060] Sequencing showed that the recombinant plasmid after inserting the DNA fragment shown in sequence 2 between the restriction sites BamH I and Not I of the pET-28a(+) plasmid was named pET-28a-UGT5.

[0061] 2. Construction of expression host system

[0062] Transform the recombinant expression vector pET-28a-UGT5 constructed in step 1 into Escherichia coli Transetta (DE3) competent cells, select positive clones for PCR verification, and send them for sequencin...

Embodiment 3

[0073] Example 3, Verification of the Activity of Rowan Phytoalexin Glycosyltransferase

[0074] 1. Experimental method

[0075] 1. Screening of catalytic activity of exogenously expressed glycosyltransferases

[0076] In the preliminary screening of glycosyltransferase activity, the crude enzyme solution of SaUGT5 was used for screening experiments. Rhaphiolepsin (compound 1, as shown in formula I), 2'-Hydroxyaucuparin (compound 2, as shown in formula II) and Noraucuparin (compound 3, as shown in formula III) are the sugar groups UDP-glucose is used as the donor for the enzymatic reaction.

[0077]

[0078] The reaction system for screening glycosyltransferase activity was: SaUGT5 crude enzyme solution (ie the supernatant in step 3(h) of Example 2) 194 μL; UDP-glucose (40 mM) 4 μL; acceptor substrate (40 mM) 2 μL.

[0079] In the experiment, the empty control supernatant prepared in Example 2 was used as a control.

[0080] The reaction conditions for glycosyltransfera...

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Abstract

The invention discloses a sorbus-aucuparia-derived phytoalexin glycosyl transferase and an encoding gene and application. The protein provided by the invention is protein a) or b) or c) as follows: the protein a) is a protein shown in a sequence 1; the protein b) is a protein obtained by replacement and / or deletion and / or addition of one or a plurality of amino acid residues of the sequence 1, and the protein b) is sourced from sorbus aucuparia and derived from the sequence 1 and has phytoalexin glycosyltransferase activity; and the protein c) is 99%, 95%, 90%, 85% or 80% or more-homologous to the qualified sequence of the protein a) or the protein b), the protein c) is derived from the sorbus aucuparia and has phytoalexin glycosyltransferase activity. The protein has the phytoalexin glycosyltransferase activity, and can be used for glycosylation of hydroxyl at different positions of biphenyl phytoalexin so as to further obtain various glycoside compounds. The invention provides a method for synthesizing biphenyl glucoside phytoalexin which has great significance for the study of disease resistance of a subfamily plant of apple and breeding of a resistant variety.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a phytoalexin glycosyltransferase derived from Sorbus chinensis and its coding gene and application. Background technique [0002] Plants produce a large number of small molecular organic compounds that are not necessary for growth and development through secondary metabolism. There are various types and different chemical structures. It is now known that there are about 100,000 secondary metabolites, including glycosides, terpenes, phenols, flavonoids, coumarins, lignans, alkaloids, steroids, saponins, polyynes, organic acids, etc., many Secondary metabolites have important pharmacological activity and economic value. Secondary metabolites are modified after the basic skeleton of the structure is formed, such as hydroxylation, methylation, acylation or combination of small molecules, and finally generate various end products. Glycosylation is not only a widely existing compound modi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/44
CPCC12N9/1048C12P19/44
Inventor 黄璐琦周良云郭兰萍李佳兴王升蒋靖怡刘亚辉
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI