热激蛋白基因CfHsp70-1和CfHsp70-2在热驯化增强锈赤扁谷盗高温耐受性中的作用

doi:10.3864/j.issn.0578-1752.2025.05.008

摘要/Abstract

摘要：

【目的】热激蛋白是生物体重要的分子伴侣，在生物抵御外界不良环境胁迫中具有重要作用。锈赤扁谷盗（Cryptolestes ferrugineus）是一种世界性储粮害虫，拥有极强的环境适应性。论文旨在揭示热激蛋白基因CfHsp70-1和CfHsp70-2在该害虫高温耐受性形成过程中的作用。【方法】分别利用亚致死高温（37和42 ℃）对锈赤扁谷盗驯化2 h，检测试虫对致死高温（50 ℃）耐受性变化情况。基于转录组数据鉴定获得两个锈赤扁谷盗关键热激蛋白基因（CfHsp70-1和CfHsp70-2），开展氨基酸序列和系统发育树分析。利用实时荧光定量PCR技术解析CfHsp70-1和CfHsp70-2对亚致死高温胁迫响应的表达模式。使用RNA干扰（RNA interference，RNAi）技术分别沉默CfHsp70-1和CfHsp70-2，分析不同条件下锈赤扁谷盗高温耐受性变化情况。【结果】锈赤扁谷盗经亚致死高温驯化2 h后，不同种群试虫在致死高温（50 ℃）条件下的半致死时间（LT₅₀）显著增加，表明其高温耐受性得到显著提升。序列和进化树分析显示，CfHsp70-1和CfHsp70-2氨基酸序列均拥有3个保守的Hsp70家族特征序列，且与其他鞘翅目昆虫Hsp70蛋白聚为一支。实时荧光定量PCR结果显示，锈赤扁谷盗经37和42 ℃驯化2 h，热激蛋白基因CfHsp70-1和CfHsp70-2均可被显著诱导表达。通过RNAi技术分别有效沉默CfHsp70-1和CfHsp70-2后，锈赤扁谷盗高温耐受性显著下降，即试虫在50 ℃条件下的死亡率均显著增加。【结论】热激蛋白基因CfHsp70-1和CfHsp70-2在热驯化增强锈赤扁谷盗高温耐受性中发挥重要作用。

关键词: 锈赤扁谷盗, 热驯化, 热激蛋白, 高温耐受性, RNA干扰

Abstract:

【Objective】Heat shock proteins (Hsps) are essential molecular chaperones in organisms and play the crucial roles in resisting adverse environmental stresses. Cryptolestes ferrugineus is a cosmopolitan pest of stored grains with strong environmental adaptability. This study aims to elucidate the crucial roles of heat shock protein genes CfHsp70-1 and CfHsp70-2 in the development of high-temperature tolerance in this pest.【Method】C. ferrugineus was acclimated to sub-lethal temperatures (37 and 42 ℃) for 2 h to examine the tolerance changes to lethal high temperature (50 ℃). Two key heat shock protein genes (CfHsp70-1 and CfHsp70-2) were identified based on the transcriptome data of C. ferrugineus, and the amino acid sequences and phylogenetic analysis of Hsp70 proteins were further conducted. The quantitative real-time PCR method was employed to analyze the expression patterns of CfHsp70-1 and CfHsp70-2 in response to sub-lethal heat stress. The RNA interference (RNAi) technology was used to silence CfHsp70-1 and CfHsp70-2, and then the changes in high-temperature tolerance of C. ferrugineus under different conditions were analyzed.【Result】The median lethal time (LT₅₀) of different C. ferrugineus populations was significantly increased under lethal heat temperature conditions (50 ℃) after acclimation of insects to sub-lethal temperatures (37 and 42 ℃) for 2 h, indicating a substantial enhancement of the high-temperature tolerance. The further sequence and phylogenetic analysis revealed that the amino acid sequences of CfHsp70-1 and CfHsp70-2 contained three conserved Hsp70 family signature motifs, and they clustered together with Hsp70 proteins of other Coleoptera insects. The results of quantitative real-time PCR analysis suggested that the expression levels of heat shock protein genes CfHsp70-1 and CfHsp70-2 were significantly up-regulated after acclimation to 37 and 42 ℃ for 2 h in C. ferrugineus. The gene functional analysis revealed that the high-temperature tolerance of C. ferrugineus was significantly reduced after the effective silencing of CfHsp70-1 and CfHsp70-2 via RNAi, that is, the mortality of the test insects at 50 ℃ increased significantly.【Conclusion】The heat shock protein genes CfHsp70-1 and CfHsp70-2 are involved in enhancing the high-temperature tolerance after heat acclimation of C. ferrugineus.

Key words: Cryptolestes ferrugineus, heat acclimation, heat shock protein (Hsp), high-temperature tolerance, RNA interference (RNAi)

陈二虎, 唐静杰, 胡顺杰, 唐培安. 热激蛋白基因CfHsp70-1和CfHsp70-2在热驯化增强锈赤扁谷盗高温耐受性中的作用[J]. 中国农业科学, 2025, 58(5): 918-928.

CHEN ErHu, TANG JingJie, HU ShunJie, TANG PeiAn. The Roles of Heat Shock Protein Genes CfHsp70-1 and CfHsp70-2 in Enhancing the High-Temperature Tolerance after Heat Acclimation in Cryptolestes ferrugineus[J]. Scientia Agricultura Sinica, 2025, 58(5): 918-928.

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引物类型Primer type	引物名称Primer name	引物序列Primer sequence (5′ to 3′)
RT-qPCR引物 Primers for RT-qPCR	CfHsp70-1-F	CACCGTGCCCGCATACTTT
	CfHsp70-1-R	CCGCCGCTGTTGGTTCGTT
	CfHsp70-2-F	TGTGCTATTGGTGGATGTA
	CfHsp70-2-R	GGTGACTGCTGGTTGA
	CfRPS13-F	ATCCGTAAGCATTTGGAACG
	CfRPS13-R	AGCCACTAAGGCTGAAGCTG
dsRNA引物 Primers for dsRNA	dsCfHsp70-1-F	ggatcctaatacgactcactataggATGTCCTCTTGGTTGATGT
	dsCfHsp70-1-R	ggatcctaatacgactcactataggGTCGTTCTTGATGGTGATG
	dsCfHsp70-2-F	ggatcctaatacgactcactataggGGCTCTGGCTTATGGT
	dsCfHsp70-2-R	ggatcctaatacgactcactataggCTGGCTCTGCTGACTT
	dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
	dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA

种群 Population	热驯化 Heat acclimation (℃)	驯化时间 Acclimation time (h)	LT₅₀after exposure for 50 ℃ (95%置信区间) (min)	回归方程 Regression equation (y=)	卡方值 χ²
江苏 Jiangsu (JS)	30 (control)	2	5.45 (4.99-5.84)	0.13x-0.22	0.92
	37	2	6.50 (6.14-6.81)	0.13x-0.35	0.95
	42	2	13.25 (12.83-13.86)	0.13x-1.25	0.90
湖南 Hunan (HN)	30 (control) (control)	2	17.51 (16.39-18.79)	0.071x-0.75	0.92
	37	2	22.78 (22.02-23.67)	0.06x-0.09	0.91
	42	2	29.36 (28.32-30.93)	0.063x-1.36	0.92