关键词: 麦红吸浆虫')">麦红吸浆虫, Hsp40, 基因克隆')">基因克隆, 滞育')">滞育, 温度胁迫')">温度胁迫, 实时定量PCR')">实时定量PCR

Abstract: 【Aim】 The orange wheat blossom midge, Sitodiplosis mosellana, escapes temperature extremes during hot summer and cold winter by undergoing obligatory larval diapause. This study aims to explore the potential roles of heat shock protein 40 gene (Hsp40) during diapause of S. mosellana. 【Methods】 The full-length cDNA of Hsp40 was cloned from S. mosellana larvae at pre-diapause stage by using RT-PCR and RACE technologies, and its nucleotide and amino acid sequences were subjected to bioinformatic analysis. The mRNA expression level of Hsp40 inpre-diapause, diapause and post-diapause larvae of S. mosellana, over-summering diapausing larvae exposed to short-term heat stress (35-50℃, ≤120 min) and over-wintering diapausing larvae exposed to short-term cold stress (0--15℃, ≤120 min) were detected with real-time quantitative PCR. 【Results】 The full-length cDNA sequence of Hsp40 was obtained from S. mosellana, and named SmHsp40 (GenBank accession number: MH001167), which is 1 553 bp in length and contains a 1 074 bp ORF encoding a protein of 357 amino acids with a predicted molecular weight of 40.87 kD and a theoretical pI of 5.31. The deduced amino acid sequence contains the conserved J-domain and HPD motif of Hsp40 protein family. Multiple sequence alignment and phylogenetic analysis indicated that SmHsp40 was most similar and closely related to Hsp40s from the Nematocera including Anopheles gambiae. The expression of SmHsp40 differed significantly among different diapause stages, and was up-regulated during the diapause period, with significantly higher expression level during summer (July to August) and winter (December to January of the following year). The expression of SmHsp40 was induced in over-summering diapausing larvae exposed to heat stress (40℃for 1 h) or over-wintering diapausing larvae exposed to cold stress (-10℃for 1 h) as compared to the untreated control, but not induced significantly under extreme temperatures higher than45℃or lower than-15℃. 【Conclusion】 SmHsp40 is involved in diapause process of S. mosellana and may contribute to diapause maintenance as well as heat and cold tolerance during diapause.

Key words: Sitodiplosis mosellana, Hsp40, gene cloning, diapause, temperature stress, -time quantitative PCR')">real-time quantitative PCR