Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses

The wheat blossom midge Sitodiplosis mosellana (Diptera: Cecidomyiidae), one of the most important wheat pests, undergoes obligatory larval diapause to survive adverse temperature extremes during hot summers and cold winters. This study aims to explore the potential roles of small heat shock protein (sHsp) gene Hsp21.9 in diapause process of S. mosellana.

RACE and RT-PCR technologies were used to clone the full-length cDNA of Hsp21.9 from S. mosellana pre-diapause larvae. Bioinformatics programs were used to characterize the nucleotide and amino acid sequence of cloned Hsp21.9. Real-time quantitative PCR (RT-qPCR) was used to determine the mRNA expression level of Hsp21.9 in pre-diapause, diapause, post-diapause quiescent and developing larvae of S. mosellana, as well as over-summering larvae exposed to short-term (≤120 min) heat stress (35-50℃) and over-wintering larvae exposed to short-term (≤120 min) cold stress (0 to -15℃). The recombinant Hsp21.9 protein was expressed by Escherichia coli prokaryotic expression technology, and then purified. The activity of bacterially expressed recombinant proteins to suppress thermal aggregation of pig heart mitochondrial malate dehydrogenase (MDH) was determined by colorimetry.

The full-length cDNA of S. mosellana Hsp21.9 (SmHsp21.9) obtained was 1 087 bp (GenBank accession number: KT749988), which contained a 582 bp open reading frame (ORF). The predicted ORF encoded a protein of 193 amino acids of which the content of glutamic acid (12.4%) was the most, and the content of cysteine (0.5%) was the least. The estimated molecular weight and isoelectric point were 21.9 kD and 5.67, respectively. The amino acid sequence of SmHsp21.9 contains typical α-crystallin domain of the sHsp family. The domain consists of six β-sheets, which forms a β-sandwich structure. Sequence alignment and phylogenetic analysis suggested that SmHsp21.9 displayed the highest amino acid identity and the closest relationship to Hsp27 from the Nematocera Chironomus riparius. RT-qPCR indicated that SmHsp21.9 expression differed significantly among different diapause stages. The expression level was decreased after the initiation of diapause, gradually increased in October, and peaked in early-to-mid phase of post-diapause (December and January). Compared with the untreated control, the expression level of SmHsp21.9 was significantly induced in over-summering larvae exposed to heat stress (35-45℃) or over-wintering larvae exposed to cold stressed (-5 to -10℃), but temperature extremes i.e. as high as 50℃ or as low as -15℃ failed to do so. The treatment duration also affected transcript levels of SmHsp21.9, with the maximum value at 30-60 min. Recombinant SmHsp21.9 proteins obtained significantly prevented heat-induced (43℃) aggregation of MDH, suggesting its significant molecular chaperone functionality.

The expression of SmHsp21.9 is regulated not only by diapause development, but also by environmental temperature. SmHsp21.9 might be involved in initiation and termination of diapause, and heat/cold tolerance during diapause in S. mosellana.