Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase
Introduction
Malate dehydrogenase (MDH, EC 1.1.1.37) catalyzes the reversible interconversion of malate and oxaloacetate (OAA) using either NADH or NADPH as coenzyme (Fig. 1) [1], playing a major role in cellular metabolic pathways such as tricarboxylic acid cycle and malate-aspartate shuttle. MDH can be founded in animals, plants and many prokaryotes [2]. MDH is normally encoded by a multi-gene family. NAD-dependent MDH is mainly assembled as dimer with subunit molecular weight between 32 and 37 kDa, whereas NADP-dependent MDH (EC 1.1.1.82) is possessed as a bigger subunit molecular weight around 42 kDa [3].
In plant, NAD-dependent MDH isoforms are discovered in cytosol, mitochondria, glyoxysome, peroxisome, and chloroplast [[4], [5], [6], [7]], and NADP-dependent MDH isoforms are mainly localized in chloroplast [6,8,9]. Plant mitochondria NAD-dependent MDH is involved in tricarboxylic acid cycle as well as photorespiratory [10]. In Arabidopsis, the cytosolic NAD-dependent MDH plays a self-protection role to against oxidative stress [7]. In cucumber, glyoxysome and peroxisome NAD-dependent MDH proteins are encoded by a single microbody gene [4]. NAD-dependent and NADP-dependent MDH are both existed in chloroplast [6,[11], [12], [13], [14]]. Plastidal NAD-dependent MDH is responsible for producing vitamin B6 to negatively regulate saline stress response [15] and maintaining energy homeostasis in Arabidopsis seeds developing [16]. Overexpression of Arabidopsis plastidal NADP-dependent MDH increases tolerance to salinity stress [6]. In alfalfa, nodule specific MDH is involved in enhancing the formation of nodule [17], and overexpression of MDH increases tolerance to aluminum by enhancing organic acid synthesis [18].
Bambusa oldhamii, also known as green bamboo, is mainly cultivated in the southeastern Asia [[19], [20], [21]]. Bamboo is noted for its fast-growth property which is controlled by plant hormones [22,23]. A genomic project has conducted in timber moss bamboo (Phyllostachys heterocycla) [24]. Although MDH is a wildly studied enzyme in higher plants, its function is totally unknown in bamboo species. To better understand the enzymatic characteristic of MDH in bamboo, we first cloned a BoMDH gene and heterologously expressed in Escherichia coli. Biochemical features of recombinant BoMDH can provide basic knowledge for further applications.
Section snippets
Plant material
Fresh green bamboo shoot was harvested during summer time from the mountain area of Mucha, Taipei City, Taiwan. Etiolated bamboo shoots were separated into shell and shoot portions, freezing in liquid nitrogen followed by storage at −80 °C before use.
Reagents
Bradford protein reagent [25], iProof DNA polymerase, molecular mass protein standards, and reagents for protein electrophoresis were purchased from Bio-Rad, USA. Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs,
Cloning of the BoMDH gene
A Lambda ZAP II library constructed from bamboo shoot cDNA was screened with a BoMDH-specific DIG labeled probe (Fig. S1) under general procedures [19]. Phagemid DNA contained the BoMDH gene was in vivo excised from positive clone and confirmed by DNA sequencing. BoMDH contained a 1077 bp ORF and encoded a 358-amino acids polypeptide or a 37 kDa protein. The full length BoMDH cDNA sequence was deposited at GenBank (NCBI, USA) with the accession number FJ715636. Protein sequence alignment
Conclusion
In this study, we identified a malate dehydrogenase gene, BoMDH, from green bamboo by screening cDNA library. The coding sequence of BoMDH was 1077 bp in size and contained no intron structure. E. coli expressed BoMDH was functioned as homodimer. The recombinant BoMDH was catalytically active, showing similar biochemical properties with MDH proteins reported in other monocot plants.
CRediT authorship contribution statement
Che-Jen Hsiao: Conceptualization, Data curation, Methodology, Formal analysis, Writing - original draft. Chun-Yen Hsieh: Conceptualization, Formal analysis, Writing - review & editing, Project administration, Funding acquisition. Lu-Sheng Hsieh: Conceptualization, Data curation, Formal analysis, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare no conflicts of interest with the contents of this article.
Acknowledgments
This study was supported, in part or in total, by a research grants from the Ministry of Science and Technology (MOST 107-2313-B-029-001-MY2, to L.S.H.), and form the Shin Kong Wu Ho-Su Memorial Hospital (2019SKHAND009, to C.Y.H), Taiwan. We thank Drs. Ping-Du Lee and Chien-Chih Yang for the helps during this study.
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