Application of Arabidopsis thaliana glycosyltransferase UGT75D1 in catalytic synthesis of auxin sugar ester

A technology of auxin sugar ester and Arabidopsis sugar, applied in fermentation and other directions, can solve problems such as reports on the application of glycosyltransferase UGT75D1 that have not yet been found, and achieve the effects of low efficiency and huge economic benefits

Inactive Publication Date: 2014-03-05
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the application of glycosyltransferase UGT75D1 in catalyzing the synthesis of auxin sugar esters

Method used

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  • Application of Arabidopsis thaliana glycosyltransferase UGT75D1 in catalytic synthesis of auxin sugar ester
  • Application of Arabidopsis thaliana glycosyltransferase UGT75D1 in catalytic synthesis of auxin sugar ester
  • Application of Arabidopsis thaliana glycosyltransferase UGT75D1 in catalytic synthesis of auxin sugar ester

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Embodiment 1

[0027] Example 1 Cloning, prokaryotic expression and enzyme protein purification of Arabidopsis thaliana glycosyltransferase gene UGT75D1

[0028] 1. Cloning of Arabidopsis thaliana glycosyltransferase gene UGT75D1

[0029] The glycosyltransferase gene UGT75D1 involved in the present invention is cloned from Arabidopsis thaliana by RT-PCR amplification technology. Firstly, RNA was extracted from the young leaves of Arabidopsis thaliana by TRIzol method, and then the coding region cDNA of the gene was amplified by RT-PCR method. A pair of primers used for amplification are:

[0030] UGT75D1-a: 5'-CAGGATCATGGCCAACAACAATTCCAACT';

[0031] UGT75D1-b: 5'-GCGTCGACTCACATGTGCTCATCGACAAAAG-3'.

[0032] The RT-PCR amplification program is: 94°C (pre-denaturation), 5min; 94°C (denaturation), 10s; 55°C (annealing), 15s; 72°C (extension), 2min; 35cycle; 72°C (final extension), 10min. The amplified product is recovered and purified. The amplified target gene UGT75D1 was connected with...

Embodiment 2

[0041] Example 2 Arabidopsis thaliana glycosyltransferase UGT75D1 catalyzes the synthesis of auxin sugar esters

[0042] 1. Enzyme-catalyzed reaction of auxin

[0043] The fusion protein of the purified Arabidopsis thaliana glycosyltransferase UGT75D1 (GST-UGT75D1, which can reflect the catalytic activity of UGT75D1, which is a common practice) was used for in vitro enzyme-catalyzed reactions, and six auxin compounds were selected as substrates, including Indole propionic acid (IPA), indole butyric acid (IBA), naphthaleneacetic acid (NAA). UDP-glucose was used as the sugar donor in the catalytic reaction. The enzymatic reaction system is as follows:

[0044]

[0045] Put the above mixed reaction system in a constant temperature water bath at 37°C and react for 3 hours. The reaction tubes were snap-frozen with liquid nitrogen and stored at -20°C until HPLC analysis.

[0046] 2. HPLC identification of enzyme-catalyzed products

[0047] The above reaction mixture system w...

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Abstract

The invention discloses application of Arabidopsis thaliana glycosyltransferase UGT75D1 in catalytic synthesis of auxin sugar ester, wherein the amino acid sequence of the glycosyltransferase is disclosed as SEQ ID NO.2. The auxin is indolebutyric acid (IBA), indole propionic acid (IPA) or naphthylacetic acid (NAA). The glycosyltransferase UGT75D1 and any auxin are put in a reaction tube to perform enzymatic reaction, thereby catalytically synthesizing the corresponding auxin sugar ester. The invention provides a feasible approach for efficiently and specifically synthesizing the auxin sugar ester by using a new enzyme, and can bring in great economic benefits for auxin sugar ester synthesis industry after implementation.

Description

technical field [0001] The invention relates to the application of a glycosyltransferase, in particular to the application of an Arabidopsis thaliana glycosyltransferase UGT75D1 in catalyzing the synthesis of auxin sugar ester; it belongs to the field of biotechnology. Background technique [0002] Auxin is one of the important hormones of plants, which plays an important role in regulating and controlling the growth and development of plants. Naturally occurring auxins such as indole acetic acid (IAA), indole butyric acid (IBA), and synthetic auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA ) are widely used in scientific research and agricultural production. These auxins all contain a carboxyl group (-COOH). In plants, the hydroxyl group on the carboxyl group can be covalently bonded with glucose to form sugar esters of auxin. Sugar esters of auxin are considered to be inactive and storage forms of auxin (Bartel, 1997). At present, su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/58C12P19/18
Inventor 侯丙凯张桂芝
Owner SHANDONG UNIV
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