miR-6497-x在调控小菜蛾对真菌感染反应中的作用

doi:10.3864/j.issn.0578-1752.2025.08.007

摘要/Abstract

摘要：

【背景】酚氧化酶（PO）激活系统在昆虫先天免疫中发挥着关键作用，特别是在病原体防御方面。该系统的核心组分是酚氧化酶原激活蛋白酶（PAP），它能够直接激活酚氧化酶原（proPO）。尽管这些组分在昆虫的PO激活系统中具有重要意义，但相关研究仍然较为有限。【目的】探究微小RNA（microRNA，miRNA）在小菜蛾（Plutella xylostella）免疫金龟子绿僵菌（Metarhizium anisopliae）侵染的酚氧化酶激活系统中的调控作用，为害虫防治提供新的靶点和途径。【方法】通过生物信息学方法筛选并鉴定miRNA及其靶向的mRNA。利用实时荧光定量PCR（qRT-PCR）技术，检测miRNA、PAP2和PAP3在小菜蛾感染绿僵菌（1×10⁶ CFU/mL）后不同时间的转录水平。采用体外双荧光素酶系统测定miRNA对PAP2和PAP3的调控作用。将miRNA模拟物（mimic）或抑制物（inhibitor）注射到小菜蛾体内，12 h后感染绿僵菌，使用qRT-PCR检测PAP2和PAP3的表达水平，同时统计死亡数并测定PO活性。【结果】miR-6497-x、miR-8545-x、novel-m0313-3p和novel-m0592-5p靶向PAP3；novel-m0042-5p、pxy-miR-2756-3p和miR-9215-x靶向PAP2。qRT-PCR检测结果显示，在绿僵菌侵染后的24和48 h，miRNA与靶基因之间存在负调控关系。体外试验结果表明，miR-6497-x、novel-m0313-3p和novel-m0592-5p靶向PAP3，表现出负调控作用，而miR-9215-x靶向PAP2，表现出显著的负调控作用。活体注射miR-6497-x mimic后，在绿僵菌侵染的12—48 h，PAP3表达量显著下调，幼虫死亡率显著增加，PO活性显著减弱。相反，活体注射miR-6497-x抑制剂后，在绿僵菌侵染的12—48 h，PAP3表达量显著上调，幼虫死亡率显著降低，PO活性显著增强。然而，过表达或抑制miR-9215-x后，在绿僵菌侵染的12—48 h，PAP2表达量、幼虫死亡率和PO活性与对照组相比均无显著变化。【结论】筛选并鉴定了靶向PAP3的miR-6497-x。离体和活体试验证实，miR-6497-x对PAP3具有负调控作用，进而影响PO级联反应。这表明miR-6497-x在调控小菜蛾对金龟子绿僵菌感染的免疫防御中发挥了重要作用，为针对害虫免疫系统的生物防治策略提供了理论依据。

关键词: 小菜蛾, 非编码RNA, 酚氧化酶, 昆虫免疫, 金龟子绿僵菌

Abstract:

【Background】 The phenoloxidase (PO) activation system is essential for insect innate immunity, particularly in pathogen defense, with prophenoloxidase-activating protease (PAP) being a key component that directly activates prophenoloxidase (proPO). However, research on these components in the insect PO activation system remains limited.【Objective】 The objectives of this study are to explore the regulatory role of microRNA (miRNA) in the phenoloxidase activation system of Plutella xylostella infected by Metarhizium anisopliae, and to provide new targets and approaches for pest control.【Method】 Bioinformatics was used to identify miRNAs targeting specific mRNAs. Real-time quantitative PCR (qRT-PCR) was employed to assess the transcription levels of miRNAs, PAP2, and PAP3 of P. xylostella at different time points post-infection with M. anisopliae (1×10⁶ CFU/mL). The regulatory effects of miRNAs on PAP2 and PAP3 were evaluated using a dual-luciferase system. Moths were injected with miRNA mimics or inhibitors and infected with M. anisopliae 12 h later. The expression levels of PAP2 and PAP3 were measured by qRT-PCR, while mortality and PO activity were also assessed.【Result】 miR-6497-x, miR-8545-x, novel-m0313-3p, and novel-m0592-5p target PAP3, while novel-m0042-5p, pxy-miR-2756-3p, and miR-9215-x target PAP2. A negative regulatory relationship between miRNAs and their target genes was observed at 24 and 48 h post-infection with M. anisopliae. In vitro experiments confirmed that miR-6497-x, novel-m0313-3p, and novel-m0592-5p negatively regulated PAP3, while miR-9215-x significantly downregulated PAP2. In vivo injection of miR-6497-x mimic led to decreased PAP3 expression, increased larval mortality, and reduced PO activity within 12 to 48 h post-infection. Conversely, injection of miR-6497-x inhibitor resulted in upregulated PAP3 expression, decreased larval mortality, and increased PO activity. However, overexpression or inhibition of miR-9215-x did not significantly affect PAP2 expression, larval mortality, or PO activity compared to the control group during the same period post-infection.【Conclusion】 The miR-6497-x targeting PAP3 was screened and identified. Both in vitro and in vivo experiments confirm that miR-6497-x negatively regulates PAP3, thereby affecting the PO cascade. miR-6497-x plays a crucial role in modulating the immune defense of P. xylostella against M. anisopliae infection, which will provide a theoretical basis for biological control strategies targeting pest immune systems.

Key words: diamondback moth (Plutella xylostella), non-coding RNA, phenoloxidase (PO), insect immunity, Metarhizium anisopliae

李林, 张元珍, 鄢文英, 曾路, 庞锐, 许小霞, 金丰良. miR-6497-x在调控小菜蛾对真菌感染反应中的作用[J]. 中国农业科学, 2025, 58(8): 1550-1563.

LI Lin, ZHANG YuanZhen, YAN WenYing, ZENG Lu, PANG Rui, XU XiaoXia, JIN FengLiang. The Role of miR-6497-x in Regulating the Reaction of Plutella xylostella to Fungal Infection[J]. Scientia Agricultura Sinica, 2025, 58(8): 1550-1563.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.08.007

https://www.chinaagrisci.com/CN/Y2025/V58/I8/1550

图/表 10

表1

本研究使用的引物"

目的Purpose	引物名称Primer name	引物序列Primer sequence (5′-3′)
载体构建 Vector construction	PAP3-F	ccgctcgagcggATGACTCCGTTAATCTTAATATCTGC
	PAP3-R	aaggaaaaaagcggccgcaaaaggaaaaGCCGACCGGGCCCGCA
	PAP2-F	ccgctcgagcggCCATCTATCCGAGTTCAACACGT
	PAP2-R	aaggaaaaaagcggccgcaaaaggaaaaCAGCGGTCCCCCCGA
	psi-check2-F	GCAACTACAACGCCTACCTTCGG
	psi-check2-R	CGAAAAGGTCACACTCTGGGGCG
实时荧光定量PCR qRT-PCR	U6-F	CGCAAGGATGACACGCAA
	U6-R	GAATCGAGCACCAGTTACGC
	RPS13-F	TCAGGCTTATTCTCGTCG
	RPS13-R	GCTGTGCTGGATTCGTAC
	qPAP2-F	GAAGCCTGACCTCATCCAAGA
	qPAP2-R	ACGACACCGACCACAATCC
	qPAP3-F	GGACTGCTGAGGTTGAAGGA
	qPAP3-R	TTGGCGGTGATGTCTGATGA
	novel-m0042-5p-F	TCCGAGTCTTCAAGGAACTGTGA
	pxy-mir-2756-3p-F	CCCCCGAGTGCCTACAGCGACT
	miR-9215-x-F	GAGGACGTGTGGCAGCCA
	miR-6497-x-F	GGGTTTGGAGGGGAAGCG
	miR-8545-x-F	GCCGGGCCTGGTCGATGT
	novel-m0313-3p-F	CTCCGTGGTGGAGAGGATGGTG
	novel-m0592-5p-F	TAGAAGCTGTCAGCATCGATGGGC
	miRNA universal primers-R	TAACGAGACGACGACAGAC
mimic和inhibitor合成 mimic and inhibitor synthesis	miR-6497-x mimics sense	GGGUUUGGAGGGGAAGCG
	miR-6497-x mimics antisense	CUUCCCCUCCAAACCCUU
	miR-6497-x inhibitor	CGCUUCCCCUCCAAACCC
	miR-8545-x mimics sense	GCCGGGCCUGGUCGAUGU
	miR-8545-x mimics antisense	AUCGACCAGGCCCGGCUU
	novel-m0313-3p mimics sense	UAGAAGCUGUCAGCAUCGAUGGGC
	novel-m0313-3p mimics antisense	CCAUCGAUGCUGACAGCUUCUAUU
	novel-m0592-5p mimics sense	AACGCAGAGUGGGAUAGACU
	novel-m0592-5p mimics antisense	UCUAUCCCACUCUGCGUUUU
	novel-m0042-5p mimics sense	UCCGAGUCUUCAAGGAACUGUGA
	novel-m0042-5p mimics antisense	ACAGUUCCUUGAAGACUCGGAUU
	pxy-miR-2756-3p mimics sense	CCCCCGAGUGCCUACAGCGACU
	pxy-miR-2756-3p mimics antisense	UCGCUGUAGGCACUCGGGGGUU
	miR-9215-x mimics sense	GAGGACGUGUGGCAGCCA
	miR-9215-x mimics antisense	GCUGCCACACGUCCUCUU
	miR-9215-x inhibitor	UGGCUGCCACACGUCCUC
	miRNA inhibitor NC	CAGUACUUUUGUGUAGUACAA
	miRNA mimics NC sense	UUCUCCGAACGUGUCACGUTT
	miRNA mimics NC antisense	ACGUGACACGUUCGGAGAATT

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