Thioredoxin peroxidase gene is involved in resistance to biocontrol fungus Nomuraea rileyi in Spodoptera litura: Gene cloning, expression, localization and function

Thioredoxin peroxidases (Tpxs) are a ubiquitous family of antioxidant enzymes that play important roles in protecting organisms against oxidative stress. Here, one Tpx was cloned from Spodoptera litura named as SITpx. The full-length cDNA consists of 1165 bp with 588 bp open reading frame, encoding 195 amino acids. The putative amino acid sequence shared >70% identity with Tpxs from other insects. Phylogenetic analysis revealed that SITpx is closely related to other available lepidopteran Tpxs. Real-time PCR analysis showed that SITpx can be induced by Nomuraea rileyi infection in some detected tissues at the mRNA level. The strongest expression was found in hemocytes of unchallenged and N. ri/eyi-challenged S. litura. Western blotting showed SITpx protein in the hemocytes, head and cuticle from normal S. litura. However, when N. rileyi was inoculated into the body cavity of S. litura larvae, SlTpx protein was detected in head, hemocytes, fatbody, midgut, malpighian tubule, but not in the hemolymph and cuticle. Moreover, time-course analysis showed that SITpx mRNA/protein expression levels were up-regulated in the hemocytes, when S. litura were infected by N. rileyi or injected with H202. The levels of N. rileyiinduced reactive oxygen species (ROS) in hemocytes were evaluated, and revealed that N. rileyi infection caused generation of ROS, and induced changes in expression of SITpx. In addition, the heterologously expressed protein of this gene in Escherichia coli showed antioxidant activity; it removed H202 and protected DNA. Knocking down SlTpx transcripts by dsRNA interference resulted in accelerated insect death with N. rileyi infection. This is believed to be the first report showing that SITpx has a significant role in resisting oxidative stress caused by N. rileyi infection. (C) 2013 Elsevier Ltd. All rights reserved.