Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporters.

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2019-03-01 Epub Date: 2019-02-01 DOI:10.1002/cppb.20087

Sharon Pike, Michaela S Matthes, Paula McSteen, Walter Gassmann

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引用次数: 9

Abstract

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background-free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL-LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non-plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

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利用非洲爪蟾卵母细胞对植物转运体进行功能表征。

植物膜转运蛋白的功能表征具有挑战性。通常使用异源系统来研究它们。南非爪蟾(Xenopus laevis)的未成熟卵(卵母细胞)被认为是此类研究的理想表达系统。这些大卵母细胞的质膜内源性转运系统数量少，外源mRNA表达量高;卵母细胞质膜是缺乏公认的细胞器分选信号的整体膜蛋白的默认目的地。这些特征有助于对假定的植物膜转运蛋白进行几乎无背景的表征。本文描述了如何分离非洲爪蟾卵母细胞，以制备玉米硼进口源TASSEL-LESS1 (TLS1)的带帽RNA (cRNA)为例，将cRNA微注射到分离的卵母细胞中，并在卵母细胞肿胀实验中对TLS1的硼进口能力进行功能评估。这些协议可以很容易地适用于研究其他具有假定进口功能的植物和非植物转运蛋白。©2019 by John Wiley & Sons, Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.