环境胁迫对锈赤扁谷盗呼吸速率及线粒体编码基因表达水平的影响

doi:10.3864/j.issn.0578-1752.2023.24.006

摘要/Abstract

摘要：

【背景】线粒体是生物体内一种重要细胞器，是细胞消耗氧和产生能量物质三磷酸腺苷（ATP）的主要场所，在生物体抵抗逆境过程中发挥重要作用。锈赤扁谷盗（Cryptolestes ferrugineus）是一种世界性储粮害虫，具有极强的环境适应性。【目的】分析锈赤扁谷盗呼吸速率及线粒体编码基因对不同环境胁迫的响应，探究线粒体在锈赤扁谷盗抗逆境胁迫中的应激反应。【方法】根据锈赤扁谷盗线粒体基因组数据鉴定获得线粒体编码基因，并设计相应实时荧光定量PCR（RT-qPCR）引物。通过生物测定方法得出锈赤扁谷盗对熏蒸剂（甲酸乙酯）、植物源杀虫剂（鱼藤酮）和储粮保护剂（阿维菌素）的毒力回归方程及LC₃₀，并用该浓度对试虫进行后续药剂胁迫处理。利用RT-qPCR技术解析锈赤扁谷盗线粒体编码基因的时空（不同发育阶段和幼虫不同组织）表达模式。最后，使用CO₂检测仪和RT-qPCR技术研究锈赤扁谷盗在高温（35和40 ℃）、甲酸乙酯、鱼藤酮、阿维菌素、饥饿等逆境胁迫下呼吸速率的变化以及线粒体编码基因的表达模式。【结果】共设计12个线粒体编码基因（除nad6）的定量引物。RT-qPCR结果显示这些线粒体编码基因在3龄幼虫阶段均具有较高表达水平，且线粒体基因在3龄幼虫马氏管中特异性高表达。此外，高温胁迫下锈赤扁谷盗呼吸速率呈现显著上升趋势，线粒体编码基因nad2、cytb及cox2表达水平显著上升，而nad4、nad4L、cox3及atp6则呈现显著下调趋势。甲酸乙酯熏蒸胁迫下锈赤扁谷盗呼吸速率显著降低，12个线粒体编码基因均显著下调表达，其中nad4L和nad5的表达水平仅为对照组的3.48%和1.91%。鱼藤酮和阿维菌素胁迫下锈赤扁谷盗呼吸速率均显著降低，且除cox2外的线粒体编码基因表达量均显著下调。饥饿胁迫下锈赤扁谷盗呼吸速率显著降低，且随着胁迫时间增加，线粒体编码基因表达水平下调愈加显著。【结论】锈赤扁谷盗呼吸代谢速率以及线粒体编码基因表达水平在不同逆境条件下均发生显著变化，暗示线粒体在锈赤扁谷盗适应高温、药剂和饥饿胁迫过程中发挥重要作用。

关键词: 锈赤扁谷盗, 环境胁迫, 呼吸速率, 线粒体编码基因

Abstract:

【Background】Mitochondria is an important organelle in the organism, which is the primary site for cellular oxygen consumption and the production of the energy substance adenosine triphosphate (ATP), playing a significant role in the organism’s resistance to adversity. The rusty grain beetle (Cryptolestes ferrugineus) is a type of global stored grain pest, possessing extremely strong environmental adaptability.【Objective】The objective of this study is to analyze the respiratory rate of C. ferrugineus and the response of mitochondrial protein-coding genes to different environmental stresses, and to investigate the stress response of mitochondria in the adversity resistance of C. ferrugineus.【Method】Mitochondrial protein-coding genes were identified based on the mitochondrial genome data of C. ferrugineus, and corresponding real-time fluorescence quantitative PCR (RT-qPCR) primers were designed. The toxicity regression equation and LC₃₀ of C. ferrugineus to fumigants (ethyl formate), botanical insecticides (rotenone), and stored grain protectants (avermectin) were determined by using bioassay methods, and these concentrations were used for subsequent drug stress treatment on the test insects. The spatial and temporal expression patterns (different developmental stages and different tissues of larvae) of mitochondrial protein-coding genes in C. ferrugineus were analyzed by using RT-qPCR technology. Finally, the changes in the respiratory rate of C. ferrugineus under various adversity stresses such as high temperatures (35 and 40 ℃), ethyl formate, rotenone, avermectin, and starvation, as well as the expression patterns of mitochondrial protein-coding genes, were studied by using a CO₂ detector and RT-qPCR technology, respectively.【Result】Twelve mitochondrial protein-coding genes (excluding nad6) quantitative primers were designed. RT-qPCR results showed that these mitochondrial protein-coding genes had a higher expression level at the 3rd instar larval stage, and mitochondrial genes were specifically highly expressed in the Malpighian tubules of 3rd instar larvae. Moreover, under high-temperature stress, the respiratory rate of C. ferrugineus significantly increased, and the expression levels of mitochondrial protein-coding genes nad2, cytb, and cox2 increased significantly, while nad4, nad4L, cox3, and atp6 showed a significant downregulation trend. Under ethyl formate fumigation stress, the respiratory rate of C. ferrugineus significantly decreased, and all 12 mitochondrial protein-coding genes were significantly downregulated. Among them, the expression levels of nad4L and nad5 were only 3.48% and 1.91% of the control group, respectively. Under rotenone and avermectin stress, the respiratory rate of C. ferrugineus significantly decreased, and the expression levels of mitochondrial protein-coding genes, except for cox2, were significantly downregulated. Under starvation stress, the respiratory rate of C. ferrugineus significantly decreased, and as the stress duration increased, the downregulation of mitochondrial encoded gene expression levels became more pronounced.【Conclusion】The respiratory metabolism rate and mitochondrial protein-coding gene expressions of C. ferrugineus changed significantly under different environmental stresses, indicating that the mitochondria plays an important role in the adaption to high temperature, pesticides, and starvation stress in C. ferrugineus.

Key words: Cryptolestes ferrugineus, environmental stress, respiratory rate, mitochondrial protein-coding gene

陈二虎, 袁国庆, 孙晟源, 唐培安. 环境胁迫对锈赤扁谷盗呼吸速率及线粒体编码基因表达水平的影响[J]. 中国农业科学, 2023, 56(24): 4866-4879.

CHEH ErHu, YUAN GuoQing, SUN ShengYuan, TANG PeiAn. The Effect of Environmental Stress on Respiratory Rate and Expression Level of Mitochondrial Protein-Coding Genes in Cryptolestes ferrugineus[J]. Scientia Agricultura Sinica, 2023, 56(24): 4866-4879.

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引物名称 Primer name	引物序列 Primer sequence (5′ to 3′)	产物长度 Product length (bp)	决定系数 Determination coefficient (R²)	扩增效率 Amplification efficiency (%)
nad1-F	TAATGGGTTAGTTCAGCCTT	296	1.000	91.464
nad1-R	AAATAGTTTGAGCAACAGCC	296	1.000	91.464
nad2-F	TAGTATATGGCTAGGACTGGA	92	1.000	88.099
nad2-R	TTAAGGCTGATTCTGATGGA	92	1.000	88.099
nad3-F	TTGACCCTAAATCTACCGCA	219	0.993	102.414
nad3-R	TGGCTCAGTTTAAGGCTCCT	219	0.993	102.414
nad4-F	TGCTTATTCTTCTGTTGCTC	166	0.991	102.742
nad4-R	ACTTCGTCTATGAGTTCGTT	166	0.991	102.742
nad4L-F	TTAACTATAAGTGTATGTGAGGG	63	0.996	91.195
nad4L-R	ATAATATAATCATTTCCATGC	63	0.996	91.195
nad5-F	GCCCTTTCAACTTTAAGTCA	89	0.995	106.110
nad5-R	TAAAGCCTTAAAGAGGGCAT	89	0.995	106.110
cytb-F	GAGGTGCCACAGTTATTAC	109	0.992	102.519
cytb-R	CGAGTTAATGTTGCGTTATC	109	0.992	102.519
cox1-F	TGCTCATGGAGGATCTTCAG	122	0.994	102.676
cox1-R	TTATACCTTGGGGCCGTATA	122	0.994	102.676
cox2-F	CGTTCGTCCAATAATTGTTG	128	0.998	93.275
cox2-R	AAGTCCTTCCGATTCCAG	128	0.998	93.275
cox3-F	CGCCGTTTACTATTGCTGAT	153	0.993	103.234
cox3-R	ACCCAAAATGGTGAGTTCTT	153	0.993	103.234
atp6-F	ATATTAGCCCATTTAGTCCCACA	115	0.998	93.255
atp6-R	CAGATAGCCGGACAGCCAAA	115	0.998	93.255
atp8-F	TTCCTCAAATAGCCCCTTTAAGT	50	0.997	97.458
atp8-R	AGTAAAATAGAAGAAAAGAGTTAAT	50	0.997	97.458
RPS13-F	ATCCGTAAGCATTTGGAACG	162	0.997	91.571
RPS13-R	AGCCACTAAGGCTGAAGCTG	162	0.997	91.571
EF1α-F	CCAGGCATGGTAGTGACCTT	184	0.999	96.033
EF1α-R	TTGGAGGGTTGTTTTTGGAG	184	0.999	96.033

杀虫剂Insecticide	回归方程Regression equation (y=)	LC₃₀ (95%置信区间95% CI)	决定系数Determination coefficient (R²)
甲酸乙酯Ethyl formate	-3.665+3.409x	8.3 μL·L^-1 (6.2-10.1)	0.953
鱼藤酮Rotenone	-7.903+2.285x	1696.7 mg·L^-1 (1176.7-2138.5)	0.986
阿维菌素Avermectin	-2.007+3.640x	2.6 mg·L^-1 (1.5-3.1)	0.930