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瓜实蝇蜕皮激素合成通路Halloween基因鉴定及Shade的功能分析

引用本文：王韵,马梦,李伟军,王进军,魏冬.瓜实蝇蜕皮激素合成通路Halloween基因鉴定及Shade的功能分析.植物保护学报,2024,51(2):394-404

DOI：10.13802/j.cnki.zwbhxb.2024.2023014

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作者	单位	E-mail
王韵	西南大学植物保护学院, 昆虫学及害虫控制工程重庆市重点实验室, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715
马梦	西南大学植物保护学院, 昆虫学及害虫控制工程重庆市重点实验室, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715
李伟军	江西农业大学农学院, 南昌 330045
王进军	西南大学植物保护学院, 昆虫学及害虫控制工程重庆市重点实验室, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715
魏冬	西南大学植物保护学院, 昆虫学及害虫控制工程重庆市重点实验室, 长江上游农业生物安全与绿色生产教育部重点实验室, 重庆 400715	dongwei08@swu.edu.cn

中文摘要:为明确世界检疫性害虫瓜实蝇Zeugodacus cucurbitae蜕皮激素合成通路中Halloween基因的功能，采用反转录聚合酶链式反应（reverse transcription-polymerase chain reaction，RT-PCR）技术克隆鉴定Halloween基因，通过实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）技术分析其在瓜实蝇中的时空表达模式，并利用RNA干扰（RNA interference，RNAi）技术解析蜕皮激素合成通路基因Shade对瓜实蝇变态发育的影响。结果表明，共鉴定到5个瓜实蝇蜕皮激素合成通路相关Halloween基因，即ZcSpook（缩写为ZcSpo）、ZcPhantom（ZcPhm）、ZcDisembodied（ZcDib）、ZcShadow（ZcSad）和ZcShade（ZcShd），这5个Halloween基因均具有细胞色素P450家族特有的保守结构域；系统发育分析显示瓜实蝇Halloween基因与橘小实蝇Bactrocera dorsalis的同源基因亲缘关系较近。这5个基因主要在瓜实蝇幼虫前胸腺复合体中高表达，且在幼虫化蛹前和性成熟的成虫中表达量较高。利用RNAi沉默瓜实蝇ZcShd基因24 h和48 h后，该基因表达量分别下调80.54%和60.59%，且在蜕皮化蛹过程中有16.66%的幼虫出现不同程度的皱缩、变色表型，有33.33%的幼虫死亡，有33.33%的幼虫表现出不同程度的化蛹受阻，表明该基因参与调控瓜实蝇蜕皮过程，具有成为实蝇新型控制剂防控靶标的潜力。

中文关键词:瓜实蝇 Halloween基因细胞色素P450 蜕皮激素发育时空表达

Identification of Halloween genes involved in ecdysone synthesis and functional analysis of shade in melon fruit fly Zeugodacus cucurbitae

Author Name	Affiliation	E-mail
Wang Yun	Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River(Ministry of Education), Chongqing Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China
Ma Meng	Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River(Ministry of Education), Chongqing Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China
Li Weijun	School of Agricultural Sciences, Jiangxi Agricultural University, Nanchang 330045, Jiangxi Province, China
Wang Jinjun	Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River(Ministry of Education), Chongqing Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China
Wei Dong	Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River(Ministry of Education), Chongqing Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, China	dongwei08@swu.edu.cn

Abstract:To clarify the role of Halloween genes in the ecdysone synthesis pathway of melon fruit fly Zeugodacus cucurbitae, a globally significant quarantine pest, reverse transcription-polymerase chain reaction (RT-PCR) was employed for cloning the Halloween genes. Real-time quantitative PCR (RT-qPCR) was used to analyze their spatio-temporal expression patterns, and RNA interference (RNAi) was employed to investigate the effect of the ecdysone synthesis pathway gene Shade on the metamorphosic development of Z. cucurbitae. The results showed that five Halloween genes in the ecdysone synthesis pathway were identified: ZcSpook (abbr. ZcSpo), ZcPhantom (ZcPhm), ZcDisembodied (ZcDib), ZcShadow (ZcSad) and ZcShade (ZcShd). Amino acid sequence alignment exhibited a conserved cytochrome P450 domain in all five P450 enzymes. Phylogenetic analysis showed that these genes were closely related to homologs in Bactrocera dorsalis. These genes were highly expressed in the prothoracic gland complex in both larvae and adults before sexual maturity. Following RNAi-mediated silencing of ZcShd for 24 h and 48 h, gene expression was down-regulated by 80.54% and 60.59%, respectively. Statistical analysis of Z. cucurbitae pupation showed that 16.66% of larvae exhibited shrinkage and discoloration, 33.33% died, and 33.33% displayed various malformations during pupation, indicating their involvement in regulating ecdysis, which could be used as a new target for controlling Tephritidae insects.

keywords:melon fly Halloween genes cytochrome P450 ecdysone development spatio-temporal expression

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