线粒体编码基因ND6和ATP6介导锈赤扁谷盗低温耐受性形成的机制

doi:10.3864/j.issn.0578-1752.2024.22.008

Abstract

Abstract:

【Background】Low temperatures can induce adaptive responses in ectothermic animals, the grain pest Cryptolestes ferrugineus has exhibited a remarkable adaptability to cold environment. Mitochondrial protein-coding genes are crucial for maintaining respiration metabolism and ATP synthesis in organisms, and they play various functions in insects. However, the role of these genes in cold adaptation is poorly understood.【Objective】The purpose of this study is to elucidate the roles of mitochondrial protein-coding genes in the formation of cold tolerance of C. ferrugineus.【Method】The cold tolerance of C. ferrugineus ST and CK populations was determined at a lethal low temperature (-20 ℃). The respiration rate and ATP content of C. ferrugineus ST and CK populations were measured using a CO₂ detector and an ATP content assay kit. The relative expression levels of 13 mitochondrial protein-coding genes between the ST and CK populations were assessed by using RT-qPCR. RNA interference (RNAi) technology was employed to knock down the key mitochondrial protein-coding genes ND6 and ATP6 in C. ferrugineus, and then the expression levels of the remaining 12 mitochondrial protein-coding genes, respiration rate, ATP content, and changes in cold tolerance were explored after effective silencing of ND6 and ATP6.【Result】The cold tolerance of C. ferrugineus CK population was higher than that of the ST population, while the respiration rate and ATP content of the CK population were only 58.68% and 62.54% of those in the ST population, respectively. Additionally, the expression levels of 12 mitochondrial protein-coding genes (except ND3) in the CK population were significantly lower than those in the ST population. These results suggested a negative correlation between cold tolerance and physiological indicators (respiration rate, ATP content, and the expression levels of mitochondrial protein-coding genes). When the key mitochondrial protein-coding genes ND6 and ATP6 were effectively silenced via dsRNA feeding, the respiration rate and ATP content were significantly reduced, while cold tolerance in C. ferrugineus was significantly enhanced.【Conclusion】The mitochondrial protein-coding genes ND6 and ATP6 are involved in the formation of cold tolerance by regulating energy metabolism in C. ferrugineus.

Key words: Cryptolestes ferrugineus, cold tolerance, energy metabolism, mitochondrial protein-coding gene, RNA interference (RNAi)

YUAN GuoQing, CHEN ErHu, TANG PeiAn. The Mechanisms of Mitochondrial Protein-Coding Genes ND6 and ATP6 in Regulating Cold Tolerance of Cryptolestes ferrugineus[J].Scientia Agricultura Sinica, 2024, 57(22): 4483-4494.

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References 70

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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)	R²	扩增效率 Amplification efficiency (%)
ND1-F	TAATGGGTTAGTTCAGCCTT	296	1.000	91.464
ND1-R	AAATAGTTTGAGCAACAGCC	296	1.000	91.464
ND2-F	TAGTATATGGCTAGGACTGGA	92	1.000	88.099
ND2-R	TTAAGGCTGATTCTGATGGA	92	1.000	88.099
ND3-F	TTGACCCTAAATCTACCGCA	219	0.993	102.414
ND3-R	TGGCTCAGTTTAAGGCTCCT	219	0.993	102.414
ND4-F	TGCTTATTCTTCTGTTGCTC	166	0.991	102.742
ND4-R	ACTTCGTCTATGAGTTCGTT	166	0.991	102.742
ND4L-F	TTAACTATAAGTGTATGTGAGGG	63	0.996	91.195
ND4L-R	ATAATATAATCATTTCCATGC	63	0.996	91.195
ND5-F	GCCCTTTCAACTTTAAGTCA	89	0.995	106.11
ND5-R	TAAAGCCTTAAAGAGGGCAT	89	0.995	106.11
ND6-F	TCACCCCTTATCTTTCGGAGT	188	0.999	90.76
ND6-R	TGAAATTTTTCATTAGAGGCTACA	188	0.999	90.76
Cytb-F	GAGGTGCCACAGTTATTAC	109	0.992	102.519
Cytb-R	CGAGTTAATGTTGCGTTATC	109	0.992	102.519
COXI-F	TGCTCATGGAGGATCTTCAG	122	0.994	102.676
COXI-R	TTATACCTTGGGGCCGTATA	122	0.994	102.676
COXII-F	CGTTCGTCCAATAATTGTTG	128	0.998	93.275
COXII-R	AAGTCCTTCCGATTCCAG	128	0.998	93.275
COXIII-F	CGCCGTTTACTATTGCTGAT	153	0.993	103.234
COXIII-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

引物名称 Primer name	引物序列 Primer sequence (5′ to 3′)
dsND6-F	gatcctaatacgactcactataggACCCCTTATCTTTCGGAGTTACA
dsND6-R	ggatcctaatacgactcactataggGCGTAATGGGCCATATTTGATAT
dsATP6-F	ggatcctaatacgactcactataggTTTCCTCATTTGACCCCTCA
dsATP6-R	ggatcctaatacgactcactataggAATAGGCGGAGTTCCTTGTG
dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA

试虫种群 Population	回归方程 Regression equation (y=)	LT₅₀(min) (95%置信区间 95% CI)	R²
ST	6.695x-10.339	35.014 (24.759-43.426)	0.954
CK	8.445x-13.942	44.762 (40.762-48.638)	0.969

The Mechanisms of Mitochondrial Protein-Coding Genes ND6 and ATP6 in Regulating Cold Tolerance of Cryptolestes ferrugineus

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