小麦吸浆虫小热激蛋白基因Hsp21.9的克隆及在滞育过程与温度胁迫下的表达特性

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

【目的】小麦吸浆虫(Sitodiplosis mosellana)是最重要的小麦害虫之一,滞育使其度过酷暑和严寒。本研究旨在探讨小麦吸浆虫小热激蛋白(small heat shock protein,sHsp)基因Hsp21.9在滞育中的作用。【方法】利用RACE和RT-PCR技术从小麦吸浆虫滞育前幼虫中克隆Hsp21.9全长cDNA序列;利用生物信息学软件对其核苷酸和编码蛋白特性进行分析;采用RT-qPCR技术分析Hsp21.9在滞育进程(滞育前、滞育、滞育后静息期和滞育后发育)不同阶段幼虫及夏滞育幼虫在短期(≤120 min)极端高温(35-50℃)和越冬幼虫在短期(≤120 min)极端低温(0--15℃)胁迫下的表达模式;通过大肠杆菌原核表达系统诱导表达及纯化其编码蛋白,采用吸光值法测定重组蛋白抑制猪心苹果酸脱氢酶(malate dehydrogenase,MDH)热聚沉的能力。【结果】克隆获得了cDNA全长为1 087 bp的小麦吸浆虫Hsp21.9,命名为SmHsp21.9(GenBank登录号:KT749988),其开放阅读框长度为582 bp,编码193个氨基酸,其中谷氨酸含量最高(12.4%),半胱氨酸含量最低(0.5%);预测的蛋白分子量为21.9 kD,等电点为5.67。SmHsp21.9氨基酸序列具有小热激蛋白家族典型的α-晶体结构域,该结构域由6个β-折叠组成,其在空间上形成β-三明治结构。蛋白序列相似性和系统进化分析表明,SmHsp21.9蛋白与长角亚目昆虫摇蚊(Chironomus riparius)Hsp27的相似性最高、亲缘关系最近。RT-qPCR分析结果表明,小麦吸浆虫滞育不同时期幼虫SmHsp21.9表达量存在显著差异,进入滞育后表达量显著降低,10月后逐渐升高,滞育后静息阶段的12月和翌年1月显著高于其他季节。与未处理的对照相比,35-45℃高温、-5--10℃低温处理可显著诱导越夏、越冬幼虫SmHsp21.9的表达,以30-60 min处理诱导效果较明显;高于50℃或低于-15℃诱导效果不明显。获得的SmHsp21.9重组蛋白可显著抑制MDH在高温(43℃)下聚集,具有显著的分子伴侣活性。【结论】小麦吸浆虫SmHsp21.9的表达不仅受滞育发育影响,而且受环境温度的调控,其可能参与滞育的启动和终止,且与滞育期间的耐热和耐寒性相关。

【Objective】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.【Method】RACE and RT-PCR technologies were used to clone the full-length c DNA 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.【Result】The full-length cDNA of S. mosellana Hsp21.9(Sm Hsp21.9) 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 Sm Hsp21.9 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 Sm Hsp21.9 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 Sm Hsp21.9, with the maximum value at 30-60 min. Recombinant Sm Hsp21.9 proteins obtained significantly prevented heat-induced(43℃) aggregation of MDH, suggesting its significant molecular chaperone functionality.【Conclusion】The expression of Sm Hsp21.9 is regulated not only by diapause development, but also by environmental temperature.Sm Hsp21.9 might be involved in initiation and termination of diapause, and heat/cold tolerance during diapause in S. mosellana.