在酿酒酵母中测定克隆的植物细胞色素P450氧化酶的脱落酸8'-羟化酶活性。

Assay for abscisic acid 8'-hydroxylase activity of cloned plant cytochrome P450 oxidases in Saccharomyces cerevisiae.

作者信息

Eggels Stella, Avramova Viktoriya, Schön Chris-Carolin, Poppenberger Brigitte, Rozhon Wilfried

机构信息

Plant Breeding, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Liesel-Beckmann-Straße 2, 85354, Freising, Germany.

Biotechnology of Horticultural Crops, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Liesel-Beckmann-Straße 1, 85354, Freising, Germany.

出版信息

Anal Biochem. 2018 Jul 15;553:24-27. doi: 10.1016/j.ab.2018.05.011. Epub 2018 May 16.

DOI:10.1016/j.ab.2018.05.011

PMID:29777680

Abstract

The plant hormone abscisic acid (ABA) regulates many processes, including response to drought, seed dormancy and abscission of leaves and fruits. For maintenance of ABA homeostasis, catabolism of ABA by 8'-hydroxylation and subsequent cyclisation to phaseic acid (PA) is crucial. However, detection of ABA 8'-hydroxylation activity is tedious. We present a simple and rapid method for detection of ABA 8'-hydroxylase activity by cloning cDNAs of interest and expressing the respective protein in yeast. Upon addition of ABA, PA is formed and subsequently quantified in the yeast cell culture supernatant by heart cutting 2D-HPLC or GC-MS.

摘要

植物激素脱落酸（ABA）调控许多生理过程，包括对干旱的响应、种子休眠以及叶片和果实的脱落。对于维持ABA的动态平衡而言，通过8'-羟基化作用使ABA发生分解代谢并随后环化形成相酸（PA）至关重要。然而，检测ABA 8'-羟基化活性的过程繁琐。我们提出了一种简单快速的方法，通过克隆感兴趣的cDNA并在酵母中表达相应的蛋白质来检测ABA 8'-羟化酶活性。加入ABA后，会形成PA，随后通过中心切割二维高效液相色谱法（2D-HPLC）或气相色谱-质谱联用仪（GC-MS）对酵母细胞培养上清液中的PA进行定量分析。