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

Abstract

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)

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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References 47

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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

Mitochondrial Protein-Coding Genes Nad5, Nad6 and Atp6 are Involved in Phosphine Resistance of Cryptolestes ferrugineus

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