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Analytical method for related enzymes in biosynthesis pathway of plant secondary metabolites

A technology of substrates and compounds, applied in biochemical equipment and methods, biomaterial analysis, biological testing, etc., can solve problems such as incompatibility, heavy workload, and incomplete protein expression levels

Active Publication Date: 2018-07-31
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when studying the biosynthetic pathway of secondary metabolites in the eukaryotic system, due to the complex body state and different environmental conditions of eukaryotic organisms, the biosynthetic pathways of secondary metabolites will vary widely, and then use the same method as prokaryotic Not only is the workload huge, but it may not be applicable to protein-catalyzed reactions in complex organisms
Because RNA levels do not directly correspond to protein abundance, let alone the more important protein and enzyme activity; protein levels in cells may not correspond to enzyme activity responsible for cellular metabolism, regulation, and signaling, such as some The reaction may require the cooperation of several different proteins to jointly catalyze the reaction, and these proteins may not be found in genome transcription; moreover, since the activity of the protein is controlled by many post-translational modification events, such as phosphorylation, methylation , glycosylation, etc., the content of these modified active proteins can truly reflect the functional state of the protein under physiological conditions; therefore, it is not comprehensive to only study the expression level of the protein

Method used

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  • Analytical method for related enzymes in biosynthesis pathway of plant secondary metabolites
  • Analytical method for related enzymes in biosynthesis pathway of plant secondary metabolites
  • Analytical method for related enzymes in biosynthesis pathway of plant secondary metabolites

Examples

Experimental program
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experiment example 1

[0089] Experimental Example 1: Synthesis of Probe Molecule 1

[0090] 1. Experimental materials and reagents

[0091] Compound 1-1 was synthesized in this laboratory according to the method of reference (Angewandte Chemie International Edition, 2013, 52(33):8551-8556.); the reagents used for chemical synthesis were purchased from Sigma-Aldrich, TCI or aladdin respectively .

[0092] 2. Synthesis of probe molecule 1

[0093] The synthetic route of probe molecule 1 is as follows:

[0094]

[0095] Add steviol (30mg, 0.1mmol) in the dry flask, K 2 CO 3 (27mg, 0.2mmol), pumped nitrogen, added 2mL of anhydrous N,N-dimethylformamide (DMF), 3-(but-3-ynyl)-3-(2-iodoethyl)-3H -Diaziridine (1-1) (33mg, 0.13mmol) was dissolved in 1mL N,N-dimethylformamide, added to the reaction under ice-cooling, heated to 70°C, reacted for 6hr, thin layer chromatography (TLC) For detection, wash with 10% HCl and saturated saline, dry with anhydrous sodium sulfate, and separate by column chromat...

experiment example 2

[0100] Experimental Example 2: Extraction of Active Stevia Plant Protein

[0101] 1. Experimental materials and reagents

[0102] Stevia rebaudiana was collected from Jining City, Shandong Province, China. DEAE column material was purchased from Aogma.

[0103] Plant cell extract: 50mM HEPES (pH 7.8), 150mM sodium chloride, 5mM magnesium chloride, 1mM ethylenediaminetetraacetic acid, 10mM dithiothreitol, 1g sodium ascorbate, 10g cross-linked polyvinylpyrrolidone, 5g polyvinylpyrrolidone.

[0104] 2. Extraction of active stevia plant protein

[0105] Grind the fresh stevia leaf and stem tissue by liquid nitrogen grinding method, add 2mL of pre-cooled plant cell lysate added with protease inhibitors per 1g of stem and leaf tissue, and incubate on ice to fully dissolve the stevia protein , use ammonium sulfate gradient salting out to obtain protein precipitation, then use pre-cooled protein solution to redissolve the precipitated protein, after dialysis and desalination, use D...

experiment example 3

[0106] Experimental example 3: Verifying the catalytic activity of stevia protein

[0107] 1. Experimental materials and reagents

[0108] Steviol was synthesized by our laboratory, and product standards and reagents were purchased from Sigma, TCI, Sinopharm and Yuanye.

[0109] 2. Verification of the catalytic activity of stevia protein

[0110] Take 300 μL of the protein solution prepared in Example 2, add 1 mM steviol, 5 mM uridine diphosphate glucose (UDPG), react at 30 ° C for 4 hours, extract with n-butanol, spin dry, redissolve in methanol, LC-MS analysis, stevia Steviol monoside, steviol bioside, stevioside, and rebaudioside A are all produced.

[0111] Gel electrophoresis, staining. Figure 6 Silver staining analysis of extracted stevia protein after electrophoresis is shown. Cut off the gel blocks with a size around 50kD and perform protein spectrum analysis after in-gel digestion. Figure 8 Protein profiling after electrophoresis of extracted stevia proteins is...

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Abstract

The invention relates to an analytical method for related enzymes in a biosynthesis pathway of plant secondary metabolites. Specifically, the present invention provides a compound represented by the formula (I) L-B shown in the specification, wherein L is a linking group substituted by a click chemical functional group and / or a photoaffinity group; and B is a substrate of a functional protein. Theinvention also provides a probe molecule formed by the compound of formula (I) and a reporter group. The invention also provides a method for identifying functional proteins by use of the compound offormula (I) or the probe molecule and use of the compound of formula (I) or the probe molecule. The analytical method is capable of rapidly and specifically labeling the proteins in a functional state in complex biological samples.

Description

technical field [0001] The invention relates to a method for analyzing related enzymes in the biosynthetic pathway of plant secondary metabolites. Background technique [0002] There is no clear boundary between primary and secondary metabolism in plants. Primary metabolism provides many small molecular substances as precursors of secondary metabolic pathways, and in many cases the precursors of secondary metabolites are also used for the synthesis of primary metabolites. The biosynthetic metabolic pathways of secondary metabolites are diverse, many of which are still poorly understood. [0003] Traditional biological methods to study the biosynthetic pathway of secondary metabolites in prokaryotes are mainly through cDNA cloning sequencing and the analysis of the characteristics of expressed sequence tags (ESTs). In addition, due to the high gene homology of proteins with similar catalytic functions in prokaryotes, And it exists in clusters, which is more convenient and f...

Claims

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Application Information

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IPC IPC(8): C07D229/02C07D495/04C09K11/06C12N9/10G01N33/68G01N33/558G01N21/64
CPCC07D229/02C07D495/04C09K11/06C09K2211/1044C09K2211/1092C12N9/1048C12N15/8205G01N21/6428G01N33/558G01N33/6848G01N2021/6439G01N2570/00
Inventor 肖友利李伟超周怡青
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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