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麦红吸浆虫滞育不同阶段幼虫ATP含量、ATP酶活力和SmATP5A基因表达分析

引用本文：马倩,黄启通,任家欣,朱克岩,成卫宁.麦红吸浆虫滞育不同阶段幼虫ATP含量、ATP酶活力和SmATP5A基因表达分析.植物保护学报,2025,52(2):390-399

DOI：10.13802/j.cnki.zwbhxb.2025.2024104

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作者	单位	E-mail
马倩	西北农林科技大学植物保护学院, 植保资源与病虫害治理教育部重点实验室, 杨凌 712100
黄启通	西北农林科技大学植物保护学院, 植保资源与病虫害治理教育部重点实验室, 杨凌 712100 山东省蚕业研究所, 烟台 264002
任家欣	西北农林科技大学植物保护学院, 植保资源与病虫害治理教育部重点实验室, 杨凌 712100
朱克岩	Department of Entomology, Texas A&M University, Texas 77843, USA
成卫宁	西北农林科技大学植物保护学院, 植保资源与病虫害治理教育部重点实验室, 杨凌 712100	cwning@126.com

中文摘要:为探明麦红吸浆虫Sitodiplosis mosellana三磷酸腺苷（adenosine triphosphate，ATP）含量、ATP酶活力及其α亚基（ATPase α-subunit，ATP5A）基因SmATP5A的表达变化与其滞育的关系，采用ATP荧光检测试剂盒和酶连续反应比色法分别测定麦红吸浆虫滞育进程不同阶段幼虫的ATP含量和ATP水解酶/合成酶活力，通过逆转录PCR（reverse transcription PCR，RT-PCR）技术克隆SmATP5A基因cDNA序列并进行生物信息分析，采用实时荧光定量PCR （quantitative real-timePCR，qPCR）和Western-blot技术检测SmATP5A基因mRNA和蛋白在该虫滞育进程中的表达变化模式。结果表明：麦红吸浆虫进入滞育后幼虫的ATP含量、ATP水解酶和合成酶活力显著下降，并在整个滞育期维持较低水平，但进入滞育后静息期有所升高，发育恢复后再次升高，其中发育后期显著高于滞育和静息期。克隆获得了开放阅读框长度为1 659 bp的SmATP5A基因（GenBank登录号为PQ412453），该基因编码552个氨基酸，编码蛋白的分子量为59.6 kD；氨基酸序列分析发现SmATP5A具有ATP酶催化核心典型的核苷酸结合结构域及Walker A和Walker B基序，与同为瘿蚊科的甘蓝瘿蚊Contarinia nasturtii ATP5A的序列一致性最高、亲缘关系最近。qPCR和Western-blot分析结果表明，麦红吸浆虫滞育不同阶段幼虫SmATP5A基因的mRNA和蛋白的表达水平存在明显差异，其中在滞育前和滞育后发育阶段最高，其次为滞育后静息期，在滞育期最低，即与ATP含量和ATP酶活力变化模式基本一致。说明麦红吸浆虫ATP含量、ATP酶活力及SmATP5A基因表达的降低与其滞育启动和滞育期较低的代谢水平紧密相关，而其升高与滞育终止和恢复发育需要较多的能量供给密切相关。

中文关键词:麦红吸浆虫滞育 ATP含量 ATP酶活力基因表达

ATP content, ATPase activity and SmATP5A gene expression in red wheat blossom midge Sitodiplosis mosellana larvae at different diapause stages

Author Name	Affiliation	E-mail
Ma Qian	Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, China
Huang Qitong	Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, China Shandong Institute of Sericulture, Yantai 264002, Shandong Province, China
Ren Jiaxin	Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, China
Ren Jiaxin	Department of Entomology, Texas A&M University, Texas 77843, USA
Zhu Keyan	Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi Province, China	cwning@126.com

Abstract:To clarify the relationship between ATP content, ATPase activity and gene expression of ATPase α-subunit (ATP5A) in red wheat blossom midge Sitodiplosis mosellana and diapause development, ATP content and activities of ATP synthase and hydrolase in S. mosellana larvae at different diapause stages were respectively measured using an ATP fluorescence assay kit and enzyme continuous reaction colorimetry. The cDNA sequence of SmATP5A was cloned using reverse transcription PCR (RTPCR) technique, and analyzed by bioinformatics. Quantitative real-time PCR (qPCR) and Western-blot analysis were used to detect the mRNA and protein expression patterns of the SmATP5A gene in diapause process of S. mosellana. Results showed that ATP content and activities of ATP synthase and hydrolyase in S. mosellana larvae significantly decreased after entering diapause, remained at low levels throughout diapause, but increased after entering post-diapause quiescent period, and increased again during post-diapause development, with levels significantly higher in the later stage compared to those during diapause and post-diapause quiescence. Open reading frame (ORF) of SmATP5A obtained is 1 659 bp (GenBank accession number: PQ412453), encoding a protein of 552 amino acids with a predicted molecular weight of 59.6 kD. The amino acid sequence analysis suggested that SmATP5A contained the classical nucleotide-binding domain and Walker A and Walker B motifs of ATPase catalytic core. SmATP5A exhibited the highest amino acid identity and closest phylogenetic relationship to ATP5A from Contarinia nasturtii. qPCR and Western-blot analysis demonstrated that the mRNA and protein expression levels of SmATP5A showed significant differences in the course of diapause. Their expression levels were the highest at pre-diapause and post-diapause development stages, followed by post-diapause quiescence stage, and the lowest in diapause stage, which are highly consistent with change pattern of ATP content and ATPase activity. These results suggest that the decreases in ATP content, ATPase activity and SmATP5A expression during diapause in S. mosellana are closely associated with diapause initiation and reduced metabolism level in diapause stage, while their increases are closely linked to the elevated energy demands required for diapause termination and post-diapause development.

keywords:Sitodiplosis mosellana diapause ATP content ATPase activity gene expression

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