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

Abstract

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 SlTpx. The full-length cDNA consists of 1165bp with 588bp open reading frame, encoding 195 amino acids. The putative amino acid sequence shared >70% identity with Tpxs from other insects. Phylogenetic analysis revealed that SlTpx is closely related to other available lepidopteran Tpxs. Real-time PCR analysis showed that SlTpx 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. rileyi-challenged S. litura. Western blotting showed SlTpx 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 SlTpx mRNA/protein expression levels were up-regulated in the hemocytes, when S. litura were infected by N. rileyi or injected with H2O2. The levels of N. rileyi-induced reactive oxygen species (ROS) in hemocytes were evaluated, and revealed that N. rileyi infection caused generation of ROS, and induced changes in expression of SlTpx. In addition, the heterologously expressed protein of this gene in Escherichia coli showed antioxidant activity; it removed H2O2 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 SlTpx has a significant role in resisting oxidative stress caused by N. rileyi infection.