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| Cloning and Expression Analysis of a Stress-responsive Gene ZmGST23 from Maize (Zea mays) |
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Abstract Plant glutathione S-transferases (GSTs, E.C.2.5.1.18) are a superfamily of multifunctional enzymes which play important roles in primary metabolism, secondary metabolism, signal transduction and stress tolerance, etc. In our present study, the 669 bp full-length CDS region of ZmGST23 was cloned from an elite maize (Zea mays) inbred line F83 based on the mRNA sequence in GenBank (NM_001111524) using RT-PCR methods, which was predicted to encode a protein consisted of 222 amino acids. The deduced molecular weight of ZmGST23 was 24.84 kD, together with isoelectric point of 5.68. BLAST analysis revealed that the protein contained a C-terminal domain and an N-terminal domain, as well as glutathione binding sites and substrate binding pockets, and belonged to tau class of GSTs. Phylogenetic tree analysis showed that ZmGST23 had the closest evolutionary relationship with a Sorghum bicolor GST protein, which shared 94% identity in amino acids. Promoter analysis revealed that there were multiple putative cis-acting elements that were involved in abscisic acid responsiveness, anaerobic induction, auxin responsiveness, gibberellin responsiveness, and defense and stress responsiveness, and some MYB transcription factor binding site involved in drought-inducibility were also found in the promoter region of ZmGST23 gene. qRT-PCR analysis revealed that ZmGST23 was highly induced by abiotic stresses such as dehydration, waterlogging, salinity, abscisic acid, auxin, gibberellin, as well as extreme temperatures, interestingly, the expression level in leaves was higher than in roots. Tissue specific expression analysis showed that ZmGST23 expressed in all seven tested tissue types. However, the expression level of ZmGST23 varied with tissue types, the expression level of ZmGST23 in young buds and mature leaves were higher than that in young roots, silk, ear leaves, tassel and ear. The prokaryotic expression vector of ZmGST23, pEASY-E1-ZmGST23, was successfully constructed. The expression of fusion protein was obtained by inducing with 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) and its relative molecular weight was 30 kD, which was consistent with the theoretical value. The results in our present study revealed that ZmGST23 might serve as a potential stress response gene for the improvement of maize inbred lines and cultivars under stress conditions in breeding activity.
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Received: 14 October 2015
Published: 01 April 2016
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