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Molecular cloning and functional characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from Spartina alterniflora reveals its involvement in salt stress response

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Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been emerging to be a multifunctional protein involved in various cellular processes, in addition to its role in energy metabolism. In this study, the SaGAPDH gene was cloned from Spartina alterniflora based on the full-length cDNA library. The open reading frame of SaGAPDH was 1014 bp, and it was encoding 337 amino acids with a calculated molecular mass of 36.40 kDa. Multiple sequence alignment showed that SaGAPDH had high amino acid sequence identity with other plant GAPDHs, and phylogenetic analysis demonstrated that SaGAPDH had a closer affinity to GAPDH in Aeluropus lagopoides (AlGAPDH). Subcellular localization suggested that SaGAPDH was located in cytosol. The recombinant SaGAPDH protein was expressed in Escherichia coli cells to characterize its catalytic activity. And E. coli carrying SaGAPDH gene showed an increased salt stress resistance. SaGAPDH gene was induced by salt stress, and to further investigate its function, transgenic Arabidopsis plants ectopically antisense-overexpressing SaGAPDH was generated. The transgenic Arabidopsis plants showed a specific down-regulation of AtGAPC1 transcript and the GAPDH enzyme activity. They also showed decreased tolerance to salt stress and down-regulation of antioxidant enzymes including catalase, ascorbate peroxidase, superoxide dismutase, and peroxidase, as well as their transcripts. Above results were further confirmed by the aggravation of oxidative damage in SaGAPDH antisense-overexpressing transgenic Arabidopsis lines, which accumulated more reactive oxygen species (ROS) such as superoxide anion (O ·−2 ) and hydrogen peroxide (H2O2) under salt stress. This study indicated that SaGAPDH may play an important role in response to salt stress by the regulation of redox homeostasis.

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Abbreviations

ABA:

Abscisic acid

APX:

Ascorbate peroxidase

CaMV:

Cauliflower mosaic virus

CAT:

Catalase

DAB:

3,3′-Diaminobenzidine

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GFP:

Green fluorescent protein

H2O2 :

Hydrogen peroxide

IPTG:

Isopropylthio-β-galactoside

LB:

Lysogeny broth

MS:

Murashige and Skoog

NAD:

Nicotinamide adenine dinucleotide

NBT:

Nitroblue tetrazolium

NCBI:

National Center for Biotechnology Information

O ·−2 :

Superoxide anion

ORF:

Open reading frame

POD:

Peroxidase

qRT-PCR:

Quantitative reverse transcription PCR

ROS:

Reactive oxygen species

RT:

Reverse transcription

SE:

Standard error

SOD:

Superoxide dismutase

UTR:

Untranslated region

WT:

Wild type

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Acknowledgements

This work was supported by Natural Science Foundation of Jiangsu Province (BK20161380), the Jiangsu Provincial Public Institutions Program for Research Conditions and Building Capacity (BM2015019), and the Open Fund of Jiangsu Key Laboratory for Bioresources of Saline Soils (JKLBS2012010).

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  1. Huiyan Yuan and Lili Cai have contributed equally to this work.

Authors and Affiliations

  1. College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China

    Huiyan Yuan, Bing Xia & Ren Wang

  2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China

    Huiyan Yuan, Lili Cai, Peng Wang, Bin Sun, Sheng Xu, Bing Xia & Ren Wang

  3. Jiangsu Key Laboratory for Bioresources of Saline Soils, Yancheng, 224051, China

    Sheng Xu

  4. Jintanglang School of Architecture and Urban Environment, Soochow University, Suzhou, 215123, China

    Huiyan Yuan

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  1. Huiyan Yuan
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Yuan, H., Cai, L., Wang, P. et al. Molecular cloning and functional characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from Spartina alterniflora reveals its involvement in salt stress response. Acta Physiol Plant 41, 127 (2019). https://doi.org/10.1007/s11738-019-2916-8

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