miR-6497-x在调控小菜蛾对真菌感染反应中的作用  

The Role of miR-6497-x in Regulating the Reaction of Plutel a xylostella to Fungal Infection

作　　者：李林[1] 张元珍 鄢文英 曾路 庞锐 许小霞[1] 金丰良[1] LI Lin;ZHANG YuanZhen;YAN WenYing;ZENG Lu;PANG Rui;XU XiaoXia;JIN FengLiang(College of Plant Protection,South China Agricultural University/State Key Laboratory of Green Pesticide,Guangzhou 510642)

机构地区：[1]华南农业大学植物保护学院/绿色农药全国重点实验室,广州510642

出　　处：《中国农业科学》2025年第8期1550-1563,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32172498,31972345);广东省自然科学基金(2023A1515010305)。

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【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 m RNAs.Real-time quantitative PCR(qRT-PCR) was employed to assess the transcription levels of mi RNAs,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 mi RNAs 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 mi R-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 48h 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 con

关 键 词：小菜蛾 非编码RNA 酚氧化酶 昆虫免疫 金龟子绿僵菌 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]