线粒体编码基因Nad5、Nad6和Atp6参与锈赤扁谷盗磷化氢抗性形成

doi:10.3864/j.issn.0578-1752.2024.09.008

摘要/Abstract

摘要：

【背景】锈赤扁谷盗（Cryptolestes ferrugineus）是重要储粮害虫，其对磷化氢（phosphine）抗性问题尤为突出。线粒体是生物体的重要细胞器，是昆虫进行呼吸代谢反应的核心场所，其中线粒体编码基因参与调节昆虫呼吸速率、能量代谢以及细胞信号转导等生理过程。【目的】明确线粒体编码基因在锈赤扁谷盗磷化氢抗性形成过程中的作用。【方法】通过CO₂检测仪测定锈赤扁谷盗不同磷化氢抗性种群试虫的呼吸速率；利用RT-qPCR技术解析线粒体编码基因在磷化氢抗性水平和呼吸速率均差异最大的锈赤扁谷盗太仓和上海种群试虫中的表达模式，并进行线粒体复合体I和V的活性测定；通过RT-qPCR技术研究关键线粒体编码基因Nad5、Nad6和Atp6对磷化氢胁迫响应的表达模式以及胁迫后线粒体复合体I和V的活性变化。利用RNA干扰（RNA interference，RNAi）技术沉默锈赤扁谷盗线粒体编码基因Nad5、Nad6和Atp6，同时分析上述基因被有效沉默后试虫呼吸速率和磷化氢敏感性变化情况。【结果】锈赤扁谷盗呼吸速率与磷化氢抗性水平呈负相关关系，即害虫呼吸速率均随磷化氢抗性倍数增加而显著下降。RT-qPCR结果表明线粒体编码基因在锈赤扁谷盗磷化氢极高抗种群（太仓种群，RR=1 906.8）表达量显著低于相对敏感种群（上海种群，RR=1.4），且线粒体复合体I和V的酶活性与线粒体基因表达模式相一致。锈赤扁谷盗经磷化氢熏蒸胁迫后，关键线粒体编码基因Nad5、Nad6和Atp6被显著抑制，线粒体复合体I和V的酶活性均显著降低。注射dsRNA有效沉默关键线粒体编码基因Nad5、Nad6和Atp6后，锈赤扁谷盗呼吸速率显著降低，磷化氢敏感性显著下降。【结论】线粒体编码基因参与锈赤扁谷盗磷化氢抗性形成。

关键词: 锈赤扁谷盗, 储粮害虫, 磷化氢抗性, 线粒体, RNA干扰

Abstract:

【Background】Cryptolestes ferrugineus is one of the most economically important stored-grain pests, and its phosphine resistance is particularly prominent. Mitochondria are important organelles in living organisms, which are the core site for insects to undergo respiratory metabolic reactions. Mitochondrial protein-coding genes (PCGs) are involved in regulating physiological processes of insects, such as respiratory rate, energy metabolism, and cell signal transduction.【Objective】The objective of this study is to clarify the roles of mitochondrial PCGs in phosphine resistance of C. ferrugineus.【Method】The respiratory rates of different phosphine resistant populations of C. ferrugineus were measured by using a CO₂ detector. The Taicang and Shanghai populations of C. ferrugineus with greatest differences in phosphine resistance levels and respiratory rates were selected to analyze the expression patterns of mitochondrial PCGs by using RT-qPCR technology, and the activities of mitochondrial complexes I and V were measured as well. The expression levels of three key mitochondrial PCGs including Nad5, Nad6 and Atp6, and the activities of mitochondrial complex I and V were determined after phosphine fumigation treatments in C. ferrugineus. RNA interference (RNAi) was used to silence Nad5, Nad6 and Atp6, and then the changes of respiratory rate and phosphine sensitivity of C. ferrugineus were analyzed.【Result】There was a negative correlation between the respiratory rate and phosphine resistance levels of C. ferrugineus, that is, the respiratory rates significantly decreased with the increase of phosphine resistance levels. The RT-qPCR results showed that the expression levels of mitochondrial PCGs in the highly phosphine resistant population (Taicang population, RR=1 906.8) of C. ferrugineus were significantly lower than those in the relatively sensitive population (Shanghai population, RR=1.4), and the enzyme activities of mitochondrial complexes I and V were consistent with the expression patterns of mitochondrial PCGs. The expression levels of three key mitochondrial PCGs, Nad5, Nad6 and Atp6, and the activities of mitochondrial complexes I and V were significantly inhibited after phosphine fumigation treatments in C. ferrugineus. The key mitochondrial PCGs, Nad5, Nad6 and Atp6 were silenced by injecting dsRNA, which resulted in a significant decrease in respiratory rate and phosphine sensitivity of C. ferrugineus.【Conclusion】The mitochondrial PCGs are involved in phosphine resistance of C. ferrugineus.

Key words: Cryptolestes ferrugineus, stored-grain pest, phosphine resistance, mitochondria, RNA interference (RNAi)

陈二虎, 袁国庆, 陈艳, 陈梦秋, 孙晟源, 唐培安. 线粒体编码基因Nad5、Nad6和Atp6参与锈赤扁谷盗磷化氢抗性形成[J]. 中国农业科学, 2024, 57(9): 1722-1733.

CHEH ErHu, YUAN GuoQing, CHEN Yan, CHEN MengQiu, SUN ShengYuan, TANG PeiAn. Mitochondrial Protein-Coding Genes Nad5, Nad6 and Atp6 are Involved in Phosphine Resistance of Cryptolestes ferrugineus[J]. Scientia Agricultura Sinica, 2024, 57(9): 1722-1733.

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https://www.chinaagrisci.com/CN/Y2024/V57/I9/1722

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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)	扩增效率 Amplification efficiency (%)
Nad1-F	TAATGGGTTAGTTCAGCCTT	296	91.46
Nad1-R	AAATAGTTTGAGCAACAGCC	296	91.46
Nad2-F	TAGTATATGGCTAGGACTGGA	92	88.10
Nad2-R	TTAAGGCTGATTCTGATGGA	92	88.10
Nad3-F	TTGACCCTAAATCTACCGCA	219	102.41
Nad3-R	TGGCTCAGTTTAAGGCTCCT	219	102.41
Nad4-F	TGCTTATTCTTCTGTTGCTC	166	102.74
Nad4-R	ACTTCGTCTATGAGTTCGTT	166	102.74
Nad4L-F	TTAACTATAAGTGTATGTGAGGG	63	91.20
Nad4L-R	ATAATATAATCATTTCCATGC	63	91.20
Nad5-F	GCCCTTTCAACTTTAAGTCA	89	106.1
Nad5-R	TAAAGCCTTAAAGAGGGCAT	89	106.1
Nad6-F	TCACCCCTTATCTTTCGGAGT	188	90.76
Nad6-R	TGAAATTTTTCATTAGAGGCTACA	188	90.76
Cytb-F	GAGGTGCCACAGTTATTAC	109	102.52
Cytb-R	CGAGTTAATGTTGCGTTATC	109	102.52
Cox1-F	TGCTCATGGAGGATCTTCAG	122	102.68
Cox1-R	TTATACCTTGGGGCCGTATA	122	102.68
Cox2-F	CGTTCGTCCAATAATTGTTG	128	93.28
Cox2-R	AAGTCCTTCCGATTCCAG	128	93.28
Cox3-F	CGCCGTTTACTATTGCTGAT	153	103.23
Cox3-R	ACCCAAAATGGTGAGTTCTT	153	103.23
Atp6-F	ATATTAGCCCATTTAGTCCCACA	115	93.26
Atp6-R	CAGATAGCCGGACAGCCAAA	115	93.26
Atp8-F	TTCCTCAAATAGCCCCTTTAAGT	50	97.46
Atp8-R	AGTAAAATAGAAGAAAAGAGTTAAT	50	97.46
RPS13-F	ATCCGTAAGCATTTGGAACG	162	91.58
RPS13-R	AGCCACTAAGGCTGAAGCTG	162	91.58
EF1α-F	CCAGGCATGGTAGTGACCTT	184	96.03
EF1α-R	TTGGAGGGTTGTTTTTGGAG	184	96.03

引物名称 Primer name	引物序列 Primer sequence (5′ to 3′)
dsNad5-F	ggatcctaatacgactcactataggTCGTATTGGGGATGTTGCTTTAT
dsNad5-R	ggatcctaatacgactcactataggTCAAACTCAAAACAGGCTCCT
dsNad6-F	ggatcctaatacgactcactataggACCCCTTATCTTTCGGAGTTACA
dsNad6-R	ggatcctaatacgactcactataggGCGTAATGGGCCATATTTGATAT
dsAtp6-F	ggatcctaatacgactcactataggTTTCCTCATTTGACCCCTCA
dsAtp6-R	ggatcctaatacgactcactataggAATAGGCGGAGTTCCTTGTG
dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA