表皮蛋白基因TcCP14.6和TcLCPA3A参与介导赤拟谷盗对磷化氢的抗性形成

doi:10.3864/j.issn.0578-1752.2022.11.006

摘要/Abstract

摘要：

【目的】表皮蛋白是昆虫表皮重要的结构物质,大量研究已经证实表皮蛋白基因参与介导昆虫抗药性形成。以赤拟谷盗（Tribolium castaneum）为研究对象,解析表皮蛋白基因TcCP14.6（cuticle protein CP14.6）和TcLCPA3A（larval cuticle protein A3A）在昆虫磷化氢抗性形成过程中的作用。【方法】采用联合国粮农组织（FAO）推荐的磷化氢熏蒸方法对采自5个省份赤拟谷盗的磷化氢抗性水平进行测定。基于赤拟谷盗基因组数据获得TcCP14.6和TcLCPA3A的cDNA序列,利用在线生物信息学分析软件预测其编码的氨基酸序列、信号肽和保守结构域。分别提取赤拟谷盗不同组织（头、胸、腹的表皮、翅、足、肠道、马氏管和脂肪体）、不同磷化氢抗性水平以及磷化氢胁迫后试虫的总RNA。以TcRPS和TcRPL为内参基因,运用RT-qPCR技术分析TcCP14.6和TcLCPA3A在赤拟谷盗不同组织、不同磷化氢抗性水平和药剂胁迫下的表达模式。最后,通过RNA干扰技术结合生物测定方法分析TcCP14.6和TcLCPA3A与赤拟谷盗磷化氢抗性的关系。【结果】生物测定结果表明,江苏（JS,RR=1.7）和云南（YN,RR=3.0）种群对磷化氢保持敏感,湖南种群（HN,RR=20.2）表现为中等抗性,四川（SC,RR=395.4）和广东（GD, RR=862.7）种群表现为极高抗。序列分析结果表明TcCP14.6和TcLCPA3A蛋白均包含信号肽和保守的几丁质结合域。RT-qPCR结果显示,TcCP14.6和TcLCPA3A均在赤拟谷盗表皮组织中高表达,而在内部器官（脂肪体、肠道、马氏管）中的表达量相对较低。此外,TcCP14.6表达量随磷化氢抗性水平的增加呈上调趋势,而TcLCPA3A表达量则呈下降趋势;赤拟谷盗经磷化氢熏蒸处理6 h,TcCP14.6和TcLCPA3A的表达量分别呈现上调和下调趋势。注射dsRNA分别干扰抗性（GD）和敏感（YN）种群两个基因的表达后再用LC₃₀的磷化氢浓度处理试虫。与对照相比,TcCP14.6基因沉默后赤拟谷盗的死亡率显著升高,而TcLCPA3A基因沉默后试虫死亡率显著下降。【结论】表皮蛋白基因TcCP14.6和TcLCPA3A参与介导赤拟谷盗对磷化氢的抗性。

关键词: 赤拟谷盗, 磷化氢抗性, 表皮蛋白, TcCP14.6, TcLCPA3A, RNA干扰

Abstract:

【Objective】 Cuticle proteins (CPs) are the main components of insect cuticle, and numerous studies have confirmed that the CP genes were involved in insecticide resistance. The objective of this study is to clarify the roles of TcCP14.6 (cuticle protein CP14.6) and TcLCPA3A (larval cuticle protein A3A) in phosphine resistance of Tribolium castaneum.【Method】 The FAO (Food and Agriculture Organization of the United Nations)-recommended bioassay method was used to determine the phosphine resistance levels of five different T. castaneum populations. The TcCP14.6 and TcLCPA3A sequences were downloaded from the T. castaneum genome data, and their encoded amino acid sequences, signal peptides and conserved domains were predicted via the online services. The total RNAs were extracted from different tissues (the cuticles of head, thorax and abdomen, wing, leg, gut, Malpighian tubules and fat body), different phosphine resistance levels, as well as phosphine induction of T. castaneum, respectively. With TcRPS and TcRPL as internal reference genes, the RT-qPCR was used to analyze the expression patterns of TcCP14.6 and TcLCPA3A in different tissues, different phosphine resistance levels, and in response to phosphine induction. Lastly, the RNA interference (RNAi) technology and bioassay method were used to explore the relationship between the two CP genes and phosphine resistance in T. castaneum.【Result】 The bioassay analysis showed that Jiangsu (JS, RR=1.7) and Yunnan (YN, RR=3.0) belonged to susceptible populations, Hunan (HN, RR=20.2) belonged to moderately resistant population, Sichuan (SC, RR=395.4) and Guangdong (GD, RR=862.7) belonged to highly resistant populations. The sequence analysis demonstrated that both TcCP14.6 and TcLCPA3A proteins consisted of signal peptides and chitin binding domains. The RT-qPCR analysis suggested that TcCP14.6 and TcLCPA3A all had a higher expression in the peripheral tissues (the cuticles of head, thorax and abdomen, wings and legs) of T. castaneum, and with a lower expression in the internal tissues, such as fat body, gut and Malpighian tube. Besides, with the increase of phosphine resistance levels in T. castaneum, the expression level of TcCP14.6 was up-regulated, while the expression level of TcLCPA3A was down-regulated. After phosphine induction for 6 h in T. castaneum, the expression levels of TcCP14.6 and TcLCPA3A were up-regulated and down-regulated, respectively. The injection of dsRNA could significantly inhibit the expression of TcCP14.6 and TcLCPA3A in phosphine resistance (GD) and susceptible (YN) populations of T. castaneum. When treated with phosphine (LC₃₀), the mortality significantly increased after the TcCP14.6 was silenced. Instead, the mortality significantly decreased after the TcLCPA3A was silenced.【Conclusion】 The two CP genes TcCP14.6 and TcLCPA3A are involved in phosphine resistance of T. castaneum.

Key words: Tribolium castaneum, phosphine resistance, cuticle protein, TcCP14.6, TcLCPA3A, RNA interference (RNAi)

陈二虎,孟宏杰,陈艳,唐培安. 表皮蛋白基因TcCP14.6和TcLCPA3A参与介导赤拟谷盗对磷化氢的抗性形成[J]. 中国农业科学, 2022, 55(11): 2150-2160.

CHEN ErHu,MENG HongJie,CHEN Yan,TANG PeiAn. Cuticle Protein Genes TcCP14.6 and TcLCPA3A are Involved in Phosphine Resistance of Tribolium castaneum[J]. Scientia Agricultura Sinica, 2022, 55(11): 2150-2160.

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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′)
qPCR引物 Primers for qPCR	TcCP14.6-F	CGGCCATCCTCAGACTCAAC
	TcCP14.6-R	GAATTCCTGGTCGGTCCCTG
	TcLCPA3A-F	AAGGCAGCTACTCCCTCACT
	TcLCPA3A-R	GGACAACAGCGTTGAAACCG
	TcRPS18-F	CGAAGAGGTCGAGAAAATCG
	TcRPS18-R	CGTGGTCTTGGTGTGTTGAC
	TcRPL13α-F	ACCATATGACCGCAGGAAAC
	TcRPL13α-R	GGTGAATGGAGCCACTTGTT
dsRNA引物 Primers for dsRNA	dsTcCP14.6-F	ggatcctaatacgactcactataggCACCTCTCATCTCCGATGCT
	dsTcCP14.6-R	ggatcctaatacgactcactataggTTTCACTTCGCCGATCTCCT
	dsTcLCPA3A-F	ggatcctaatacgactcactataggACGAGCAACAATGGCATTCA
	dsTcLCPA3A-R	ggatcctaatacgactcactataggTATTCAACAGTACGGCGGGT
	dsGFP-F	ggatcctaatacgactcactataggATGGTGAGCAAGGGCGAGA
	dsGFP-R	ggatcctaatacgactcactataggTTACTTGTACAGCTCGTCCA

种群 Population	采集地 Collection site	回归方程 <BOLD>R</BOLD>egression equation (y=)	致死中浓度LC₅₀ (μg·L^-1) (95% CI^a)	抗性倍数 RR
JS	江苏Jiangsu	-7.108+6.009x	15.2 (14.1-16.2)	1.7
YN	云南Yunnan	-10.565+7.465x	27.2 (26.7-27.5)	3.0
HN	湖南Hunan	-6.378+2.822x	182.1 (151.6-240.7)	20.2
SC	四川Sichuan	-18.084+5.092x	3558.4 (3402.7-3726.3)	395.4
GD	广东Guangdong	-47.249+12.146x	7763.9 (7401.6-8143.6)	862.7