Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from Chilo suppressalis

Abstract

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from -11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to -3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.

Keywords: Chilo suppressalis; Expression; Temperature; Tissues; sHSPs.

Fig. 1

Nucleotide and deduced amino acid sequence of Cshsp22.9b from Chilo suppressalis. DNA extension primers and the primers used to obtain 5′/3′ sequences are underlined and arrowed from 5′ to 3′. The bold, thick horizontal line (amino acid residues 82–164) represents the conserved α-crystalline domain bordered by variable amino- and carboxy-terminal regions. The V/P/I motif is bordered with a rectangular box

Fig. 2

Nucleotide and deduced amino acid sequence of Cshsp24.3 from Chilo suppressalis. DNA extension primers and the primers used to obtain 5′/3′ sequences are underlined and arrowed from 5′ to 3′. The bold, thick horizontal line (amino acid residues 98–180) indicates the conserved α-crystalline domain; the V/P/I motif is demarcated with a rectangle

Fig. 3

Neighbor-joining phylogenetic tree of Lepidopteran sHSPs. The C. suppressalis sHSPs are bordered by rectangles. Numbers on the branches represent bootstrap values obtained from 1000 replicates (only bootstrap values >50 are shown). Abbreviations, species, and accession numbers include BmHsp23.7 (Bombyx mori, BAD74198); DpHsp23.7 (Danaus plexippus, EHJ70499); GmHsp24.8 (Grapholita molesta, AKS40086); PaHsp23.7 (Pararge aegeria, JAA83494); BmHsp20.4 (B. mori, AAG30945); AsHsp20.8 (Actias selene, ALI87024); GmHsp21.4 (G. molesta, AKS40075); BmHsp20.1 (B. mori, ACM24346); BmHsp19.9 (B. mori, BAD74195); CsHsp21.5 (Chilo suppressalis, AGM90551); Dphsp19.8 (D. plexippus, EHJ68318); CpHsp19.8 (Cydia pomonella, ADX96000); DpHsp20.4 (D. plexippus, EHJ63989); DpHsp19.7A (D. plexippus, EHJ77540); PrHsp20 (Pieris rapae, AET10424); MbHsp19.7 (Mamestra brassicae, BAF03558); CsHsp21.7B (C. suppressalis, AGM90552); SeHsp19.5 (Sesamia nonagrioides, ACD01216); PaHsp20.4 (P. aegeria, JAA83513); OpHsp22.2 (Operophtera brumata, KOB71621); CfHsp19.8 (Choristoneura fumiferana, AAZ14792); GmHsp19.8b (G. molesta, AKS40085); CsHSP19.8 (C. suppressalis, AGC23337); BmHsp20.8 (B. mori, AAG30944); SiHsp19.6 (Sesamia inferens, AJA32867); SiHsp20.6 (S. inferens, AJA32865); SnHsp20.8 (Sesamia nonagrioides, ABC68342); BmHsp22.6 (B. mori, ACM24354); PaHsp20.1B (P. aegeria, JAA78314); CfHsp21.5 (C. fumiferana, AAZ14790); DpHsp19.8B (D. plexippus, EHJ69639); DpHsp19.7B (D. plexippus, EHJ74663); CpHsp22.2 (C. pomonella, ADX96002); GmHsp22.5 (G. molesta, AKS40083); PaHsp20.1A (P. aegeria, JAA92369); DpHsp19.8A (D. plexippus, EHJ63499); CfHsp19.9 (C. fumiferana, AAZ14791); CpHsp19.9 (C. pomonella, ADX96001); GmHsp19.9 (G. molesta, AKS40077); MbHsp20.7 (M. brassicae, BAF03557); DpHsp19.8 (D. plexippus, EHJ67067); PxHsp19.5 (Plutella xylostella, BAE48744); BbHsp19.4 (Biston betularia, ADO33017); and GmHsp18.9 (G. molesta, AKS40082)

Fig. 4

Schematic representation of genomic DNA from several shsps genes. Horizontal lines and black rectangles are used to demarcate exons and introns, respectively. Cshsp, Chilo suppressalis; Bmhsp, Bombyx mori; Dmhsp, Drosophila melanogaster

Fig. 5

Relative mRNA expression levels of Cshsp22.9b and Cshsp24.3 in different tissues and organs of Chilo suppressalis. FG foregut, MG midgut, HG hindgut, FB fat body, MT Malpighian tubules, HE head, EP epidermis, HC hemocytes

Fig. 6

Relative expression levels of Cshsp22.9b in Chilo suppressalis exposed to different temperatures. a Relative expression of Cshsp22.9b at elevated temperatures (27, 30, 33, 36, 39, 42, and 43 °C). b Relative expression of Cshsp22.9b at reduced temperatures (−11, −9, −8, −6, −3, 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27 °C)

Fig. 7

Relative expression levels of Cshsp24.3 in Chilo suppressalis exposed to different temperatures. a Relative expression of Cshsp24.3 at elevated temperatures (27, 30, 33, 36, 39, 42, and 43 °C). b Relative expression of Cshsp24.3 at reduced temperatures (−11, −9, −8, −6, −3, 0, 3, 6, 9, 12, 15, 18, 21, 24, and 27 °C)