Cloning and Characterization of a Maize cDNA Encoding Glutamate Decarboxylase

Abstract

A putative glutamate decarboxylase (GAD) gene, designated ZmGAD1, was cloned from Zea mays with a combination of reverse-transcriptase polymerase chain reaction (RT-PCR) and bioinformatic approaches. The ZmGAD1 cDNA sequence contained a complete open reading frame encoding a putative protein of 496 amino acids, which contained a pyridoxal-5′-phosphate binding domain and a calmodulin (CaM)-binding domain found in nearly all GADs from plants. Sequence analysis showed that it had highest similarity with rice GAD1. Recombinant ZmGAD1 protein was expressed in Escherichia coli, purified and used to measure enzyme activity, which confirmed ZmGAD1 was really a glutamate decarboxylase gene. Southern blotting analysis suggested that ZmGAD1 was present as a single copy gene in the maize genome. RT-PCR analysis revealed that ZmGAD1 was expressed in all examined tissues including the roots, stems, leaves, ears, and tassels. The expression of the ZmGAD1 gene was upregulated and GAD activity was increased in the leaves and roots after treatment with ABA, MeJA, NaCl, PEG, or cold stress. Several stress-related ciselements were present in the ZmGAD1 promoter cloned from maize genomic DNA. These results suggested that ZmGAD1 might play an important role in responses to abiotic factors and hormone treatments.

Evaluation of candidate reference genes for qPCR in maize

“…The reference genes were chosen following the recent literature, including those newly identified by microarray data analyses present in maize atlas (Lin et al, 2009;Zhuang et al, 2010;Sekhon et al, 2011). The expression stability of these genes was subsequently assessed by using genorm PLUS (Vandesompele et al, 2002), NormFinder (Andersen et al, 2004) and BestKeeper (Pfaffl et al, 2004) algorithms.…”

Proteomics-Based Analysis of <i>Phalaenopsis amabilis</i> in Response toward <i>Cymbidium</i> Mosaic Virus and/or <i>Odontoglossum</i> Ringspot Virus Infection

“…Its expression and activity are increased in maize under stress from abscisic acid, methyl jasmonate, NaCl or cold. This up-regulation might occur at both transcriptional or post-transcriptional levels [26]. Ginseng GAD (PgGAD) gene expression is up-regulated under stress of non-biological factors such as temperature, osmotic pressure, hypoxia, and mechanical damage, and down-regulated under oxidative stress (H 2 O 2 ) [27].…”

Molecular Cloning, Sequence Analysis and Expression Analysis of an NtWRKY6 from Tobacco (<i>Nicotiana tabacum</i> L.) in Abiotic Strsss

“…Deduced anino acid sequences were aligned using Clustal W (http://npsa-pbil. ibcp.fr) software [18]. A phylogenetic tree was constructed based on full-length amino acid sequences using the neighbor-joining method [19] and was drawn using MEGA Version 4.1 [20].…”