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Application of rice gene OsIAGT1 and glycosyl transferase coded by rice gene OsIAGT1 in catalytically synthesizing auxin sugar ester

A technology of glycosyltransferase and auxin sugar ester, applied in the direction of transferase, application, genetic engineering, etc., can solve the problems of undiscovered application reports, etc., and achieve the effect of low efficiency and huge economic benefits

Inactive Publication Date: 2014-03-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there is no report on the application of the gene sequence and the encoded glycosyltransferase from rice to catalyze the synthesis of auxin sugar esters.

Method used

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  • Application of rice gene OsIAGT1 and glycosyl transferase coded by rice gene OsIAGT1 in catalytically synthesizing auxin sugar ester
  • Application of rice gene OsIAGT1 and glycosyl transferase coded by rice gene OsIAGT1 in catalytically synthesizing auxin sugar ester
  • Application of rice gene OsIAGT1 and glycosyl transferase coded by rice gene OsIAGT1 in catalytically synthesizing auxin sugar ester

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Embodiment 1

[0023] Example 1 Cloning, prokaryotic expression and enzyme protein purification of rice glycosyltransferase gene OsIAGT1

[0024] 1. Cloning of rice glycosyltransferase gene OsIAGT1

[0025] The glycosyltransferase gene OsIAGT1 involved in the present invention is cloned from rice through RT-PCR amplification technology. Firstly, the TRIzol method was used to extract the RNA from the young leaves of rice, and then the cDNA of the coding region of the gene was amplified by the RT-PCR method. The primers used for amplification were:

[0026] OsIAGT1-P1: 5'-GCCGGATCCATGGCGCATGTCCTTGTC-3';

[0027] OsIAGT1-P2: 5'-GCCCTCGAGTCACATCTCCGACGCTGC-3';

[0028] OsIAGT1-ex1:

[0029] 5'-GGGTCGACAACCCAGCCAAAACCTCGCTCTCCAGCTCGTCGAACGAGT-3';

[0030]OsIAGT1-ex2: 5'-TGGAGAGCGAGGTTTTGGCTGGGTTGTCGACCC-3', use P1 and ex1 to amplify the first exon, use P2 and ex2 to amplify the second exon, then use P1, P2 primers to amplify the two exons fusion to obtain full-length cDNA. The RT-PCR ampli...

Embodiment 2

[0039] Example 2 Rice glycosyltransferase OsIAGT1 catalyzes the synthesis of auxin sugar esters

[0040] 1. Enzyme-catalyzed reaction of auxin

[0041] The purified fusion protein of rice glycosyltransferase OsIAGT1 (GST-OsIAGT1, which can reflect the catalytic activity of OsIAGT1, which is a common practice) was used to carry out the enzyme-catalyzed reaction in vitro test tubes, and five auxin compounds were selected as substrates, including Indoleacetic acid (IAA), indolepropionic acid (IPA), indolebutyric acid (IBA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). UDP-glucose was used as the sugar donor in the catalytic reaction. The enzymatic reaction system is as follows:

[0042]

[0043]

[0044] Put the above mixed reaction system in a constant temperature water bath at 30°C and react for 3 hours. The reaction was then terminated by adding 20 μl of trichloroacetic acid at a concentration of 240 mg / ml. The reaction tubes were snap-frozen...

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Abstract

The invention discloses an application of a rice gene OsIAGT1 and glycosyl transferase coded by the rice gene OsIAGT1 in catalytically synthesizing an auxin sugar ester, wherein the nucleotide sequence of the glycosyl transferase gene OsIAGT1 is shown in SEQ ID No.1, and the amido acid sequent of the glycosyl transferase coded by the gene is shown in SEQ ID No.2. The auxin is heteroauxin, indolebutyric acid, indole-propioponic acid, naphthylacetic acid or 2,4-dichlorphenoxyacetic acid. The glycosyl transferase OsIAGT1 and any one of the auxins are put in a reaction tube for an enzymatic reaction; therefore, the corresponding auxin sugar ester can be synthesized catalytically. The invention provides a new feasible method for synthesizing the sugar esters of the auxin matters specifically at high enzymatic efficiency and; the implementation of the method brings enormous economic benefits for the auxin sugar ester synthesis industry.

Description

technical field [0001] The present invention relates to a sequence of a glycosyltransferase gene and its application, in particular to the sequence of a rice glycosyltransferase gene OsIAGT1 and the application of the encoded glycosyltransferase in catalyzing the synthesis of auxin sugar esters; technology field. Background technique [0002] Auxin was the first discovered plant hormone that regulates all aspects of plant growth and development. Whether it is synthetic auxin analogs such as 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), etc., or naturally occurring auxins such as indoleacetic acid (IAA), indolebutin Acid (IBA) is widely used in scientific research and agricultural production, and their common feature is that they all contain a carboxyl group (-COOH). In plants, 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12P19/02
Inventor 侯丙凯林继山王会勇
Owner SHANDONG UNIV
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