ABSTRACT
Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H(2)O(2), and HgCl(2). The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H(2)O(2) in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.
Subject(s)
Bees/genetics , Insect Proteins/metabolism , Peroxiredoxins/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Dithiothreitol/chemistry , Hydrogen Peroxide/toxicity , Insect Proteins/classification , Insect Proteins/genetics , Insecticides/toxicity , Mercuric Chloride/toxicity , Molecular Sequence Data , Oxidative Stress/drug effects , Peroxiredoxins/classification , Peroxiredoxins/genetics , Phylogeny , Protein Structure, Tertiary , Real-Time Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , TemperatureABSTRACT
Thioredoxin peroxidases (Tpxs), members of the antioxidant protein family, play critical roles in resisting oxidative stress. In this work, a novel 1-Cys thioredoxin peroxidase gene was isolated from Apis cerana cerana and was named AccTpx5. The open reading frame (ORF) of AccTpx5 is 663bp in length and encodes a 220-amino acid protein with a predicted molecular mass and isoelectric point of 24,921kDa and 5.45, respectively. Promoter sequence analysis of AccTpx5 revealed the presence of putative transcription factor binding sites related to early development and stress responses. Additionally, real-time quantitative PCR (Q-PCR) analysis indicated that AccTpx5 was primarily present in some developmental stages, with the highest expression levels in the first-instar larvae. The expression level of AccTpx5 was up-regulated under various abiotic stresses, including 4°C, 42°C, HgCl2, H2O2, phoxim and acaricide treatments. Conversely, it was down-regulated by UV and pyriproxyfen treatments. Moreover, H2O2 concentration dramatically increased under a variety of stressful conditions. Finally, the purified recombinant AccTpx5 protein protected the supercoiled form of plasmid DNA from damage in the thiol-dependent mixed-function oxidation (MFO) system. These results suggest that AccTpx5 most likely plays an essential role in antioxidant defence.
Subject(s)
Bees/genetics , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Amino Acid Sequence , Animals , Antioxidants/metabolism , Base Sequence , Bees/drug effects , Bees/physiology , Bees/radiation effects , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation , Insect Proteins/genetics , Insect Proteins/metabolism , Juvenile Hormones/pharmacology , Molecular Sequence Data , Open Reading Frames/genetics , Organothiophosphorus Compounds/pharmacology , Oxidative Stress , Peroxiredoxins/chemistry , Phylogeny , Protein Conformation , Pyridines/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Stress, Physiological , Ultraviolet RaysABSTRACT
BACKGROUND: As a large family of regulatory proteins, WRKY transcription factors play essential roles in the processes of adaptation to diverse environmental stresses and plant growth and development. Although several studies have investigated the role of WRKY transcription factors during these processes, the mechanisms underlying the function of WRKY members need to be further explored, and research focusing on the WRKY family in cotton crops is extremely limited. RESULTS: In the present study, a gene encoding a putative WRKY family member, GhWRKY15, was isolated from cotton. GhWRKY15 is present as a single copy gene, and a transient expression analysis indicated that GhWRKY15 was localised to the nucleus. Additionally, a group of cis-acting elements associated with the response to environmental stress and plant growth and development were detected in the promoter. Consistently, northern blot analysis showed that GhWRKY15 expression was significantly induced in cotton seedlings following fungal infection or treatment with salicylic acid, methyl jasmonate or methyl viologen. Furthermore, GhWRKY15-overexpressing tobacco exhibited more resistance to viral and fungal infections compared with wild-type tobacco. The GhWRKY15-overexpressing tobacco also exhibited increased RNA expression of several pathogen-related genes, NONEXPRESSOR OF PR1, and two genes that encode enzymes involved in ET biosynthesis. Importantly, increased activity of the antioxidant enzymes POD and APX during infection and enhanced expression of NtAPX1 and NtGPX in transgenic tobacco following methyl viologen treatment were observed. Moreover, GhWRKY15 transcription was greater in the roots and stems compared with the expression in the cotyledon of cotton, and the stems of transgenic plants displayed faster elongation at the earlier shooting stages compared with wide type tobacco. Additionally, exposure to abiotic stresses, including cold, wounding and drought, resulted in the accumulation of GhWRKY15 transcripts. CONCLUSION: Overall, our data suggest that overexpression of GhWRKY15 may contribute to the alteration of defence resistance to both viral and fungal infections, probably through regulating the ROS system via multiple signalling pathways in tobacco. It is intriguing that GhWRKY15 overexpression in tobacco affects plant growth and development, especially stem elongation. This finding suggests that the role of the WRKY proteins in disease resistance may be closely related to their function in regulating plant growth and development.
