Identification and characterization of novel ER-based hsp90 gene in the red flour beetle, Tribolium castaneum

Abstract

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Fig. 1

Alignment of the amino acid sequences of HSP90s and HSP83s. The signal peptide was marked with black box. The first 50 amino acids of HSP90 (GRP94) are distinct from the N terminus of HSP83 in both length and sequence. The ATP-bind domain located in N terminus both HSP90 and HSP83. The C-terminal conserved signature sequences of HSP83 were marked with asterisk while the HSP90 were marked with number sign. The HSP90 sequences derived from D. melanogaster (DmHSP90, CG5520); Homo sapiens (HsHSP90, CAI64497.1); T. castaneum (TcHSP90,TC012185), and the HSP83s derived from D. melanogaster (DmHSP83, CG1242); H. sapiens (HsHSP83-1,NP-005339.3; HsHSP83-2,NP-031381.2); T. castaneum (TcHSP83,TC014606)

Fig. 2

Phylogenetic analysis of HSP90s and HSP83s. Bootstrap values below 50 were removed from the tree. The ML tree is rooted by the D. melanogaster HSP60. The details of the gene information which used in the analysis were descript in Electronic supplementary material Table S1

Fig. 3

The expression pattern of Tchsp90 in different developmental stages and under cold and heat treatment. a Quantitative real-time PCR analysis of Tchsp90 mRNA relative transcript levels were normalized to ribosomal protein s3 (rps3) in the same cDNA samples. Different development stages are EE early egg (1-day-old); LE late egg (3-day-old); EL early larval (1-day-old); LL late larval (7-day-old); EP early pupae (1-day-old); LP late pupae (6-day-old); EA early adult (1-day-old); LA late adult (7-day-old). b The expression of hsp90 in cold (4 °C) and heat (45 °C) treatment. The lowercase letters were analyzed by HSD multiple compartment. Error bars represent standard deviations among three biological replications

Fig. 4

The expression pattern of Tchsp90 under starvation and the survivorship after the pupa RNAi. a Time course analysis of Tchsp90 expression under starvation stress. b The Tchsp90 expression level after RNAi. c The survivorship of n WT, IB, and ds-hsp90 beetles under starvation

Fig. 5

RNAi phenotype of Tchsp90. a RNA interference resulted in lethal phenotypes in both of larval/pupal stages. b Knocking down levels of Tchsp90 using larval RNAi. c Progressively increasing mortalities after late larval RNAi of ds-Tchsp90. d Cumulative adult mortality after late larval RNAi. Each point represented the mean days of three biological replications

Fig. 6

Reduced egg laying (a) and the egg hatch rate (b) as well as cumulative adult mortality (c) after injection of ds-Tchsp90 in 2-day-old pupae. Confidence intervals calculated using Student’s t test for 21 single pair mating and show standard deviations and significant differences compared with the control using single asterisk indicates p < 0.05; double asterisks indicates p < 0.001