Method for determining kinetic parameters of lysophosphatidic acid acyltransferase

Abstract

The invention discloses a method for determining kinetic parameters of lysophosphatidic acid acyltransferase. The method comprises the following steps: 1 transient expression of leaves and vector construction; 2 transient expression of agrobacterium strains on Benyuan leaves through agrobacterium mediation; 3 extraction of leaf cell microsomes; 4 in-vitro LPAAT reaction, and 5 LC-MS (liquid chromatography-mass spectrometry) lipomics determination. In the first step, the p19 gene is a tomato bush dwarf virus silencing suppressor, an LC-MS non-radioactive detection technology is adopted for determining the kinetic parameters of lysophosphatidic acid acyltransferase, efficient separation capacity and high-sensitivity detection capacity are achieved, different lipid types can be quantified in single analysis, the method is simple and rapid, and avoids the harm of radioactive substrates to a human body; and meanwhile, high-throughput analysis on the target lipid compound is realized by combining a triple quadrupole technology, and the dynamic range of the target type can be quantified according to factors such as the acquisition speed of a mass spectrometer and the like.

Description

technical field [0001] The invention relates to the technical field of enzyme kinetic analysis, in particular to a method for measuring kinetic parameters of lysophosphatidic acid acyltransferase. Background technique [0002] The LPAAT gene family and its homologous genes belong to the superfamily of membrane-bound acyltransferases. LPAAT is a key intermediate in the synthesis of phospholipids and triacylglycerol, and plays an important role in the production of PA. Therefore, the determination of LPAAT enzyme activity in vitro can Accurately understand the physiological functions of these enzymes. In plants, LPAAT is closely connected with chloroplasts, endoplasmic reticulum membranes and mitochondrial membranes. In Arabidopsis plastids, lysophosphatidic acid transferase is a key regulator of embryonic synthesis. Many genes are in The production of different types of lipid intermediates in different organs or tissues has great differences in function and activity. Therefor...

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Problems solved by technology

[0002] The LPAAT gene family and its homologous genes belong to the superfamily of membrane-bound acyltransferases. LPAAT is a key intermediate in the synthesis of phospholipids and triacylglycerol, and plays an important role in the production of PA. Therefore, the determination of LPAAT enzyme activity in vitro can Accurately understand the physiological functions of these enzymes. In plants, LPAAT is closely connected with chloroplasts, endoplasmic reticulum membranes and mitochondrial membranes. In Arabidopsis plastids, lysophosphatidic acid transferase is a key regulator of embryonic synthesis. Many genes are in The production of different types of lipid intermediates in different organs or tissues has great differences in function and activity. Therefore, the parameter determination, substrate preference determination and activity determination of lysophosphatidic acid acyltransferase in vitro are very meaningful, but this At present, the determination of enzymes mostly relies on radioisotope technology. The substrate of this method is high, which is not good for the human body, and the traditional method can only detect the reaction parameters of the same enzyme in the same reaction.

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