小菜蛾PxylPBAN的克隆、原核表达及其RNAi对保幼激素和性信息素通路基因表达的影响

doi:10.16380/j.kcxb.2025.05.004

昆虫学报 ›› 2025, Vol. 68 ›› Issue (5): 576-584.doi: 10.16380/j.kcxb.2025.05.004

小菜蛾PxylPBAN的克隆、原核表达及其RNAi对保幼激素和性信息素通路基因表达的影响

郑月琴^1,2, 祝潇雯³, 田厚军^1,2, 林硕^1,2, 刘倩霞¹, 魏辉^1,2,*

(1. 福建省农业科学院植物保护研究所, 农业部福州作物有害生物科学观测试验站, 福州 350013; 2. 福建省作物有害生物监测与治理重点实验室, 福州 350003; 3. 福建农林大学植物保护学院, 福州 350002)

出版日期:2025-05-20 发布日期:2025-06-06

Cloning and prokaryotic expression of PxylPBAN and its RNAi effects on the expression of genes in the juvenile hormone and sex pheromone pathways in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

ZHENG Yue-Qin^1,2， ZHU Xiao-Wen³， TIAN Hou-Jun^1,2， LIN Shuo^1,2, LIU Qian-Xia¹， WEI Hui^1,2,*

(1. Fuzhou Scientific Observing and Experimental Station of Crop Pests, Ministry of Agriculture, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2. Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou 350003, China; 3. College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

Online:2025-05-20 Published:2025-06-06

摘要/Abstract

摘要： 【目的】通过对小菜蛾Plutella xylostella性信息素生物合成激活神经肽基因PxylPBAN进行克隆、原核表达，并检测RNAi沉默PxylPBAN对小菜蛾保幼激素通路和性信息素合成通路基因表达的影响，探讨PxylPBAN在性信息素合成中的作用。【方法】利用RT-PCR克隆小菜蛾PxylPBAN的CDS全长序列；原核表达PxylPBAN并纯化；通过将dsPxylPBAN注射进小菜蛾雌蛹，利用RNAi沉默PxylPBAN，并利用RT-qPCR验证RNAi后24, 48和72 h时PxylPBAN以及保幼激素通路基因(PxylJHAMT, PxylnJHBP, PxylhJHBP和PxylMet)和性信息素合成通路基因(PxylACC和PxylFAR6)的表达量。【结果】克隆得到小菜蛾PxylPBAN(GenBank登录号：LOC105391112)，全长CDS 582 bp，编码193个氨基酸，蛋白预测分子量约为21.85 kD；原核表达获得PxylPBAN重组蛋白。RNAi结果表明与注射dsEGFP的对照比较，注射dsPxylPBAN后小菜蛾PxylPBAN的表达量显著下调，且注射后24 h时下调最显著；此外，PxylJHAMT, PxylnJHBP, PxylhJHBP, PxylMet, PxylACC和PxylFAR6的表达量也显著下调。【结论】本研究成功获得PxylPBAN重组蛋白，发现PxylPBAN是小菜蛾保幼激素通路和性信息素合成通路的重要枢纽基因，为揭示小菜蛾PxylPBAN与保幼激素对性信息素生物合成的联动调控模式奠定理论基础。

关键词: 小菜蛾, 性信息素生物合成激活神经肽, 保幼激素, 基因克隆, 原核表达, RNA干扰

Abstract: 【Aim】 This study focuses on the cloning and prokaryotic expression of the sex pheromone biosynthesis activating neuropeptide gene PxylPBAN in the diamondback moth, Plutella xylostella. Additionally, the effects of RNAi-mediated PxylPBAN silencing on the expression of genes involved in both the juvenile hormone pathway and the sex pheromone synthesis pathway were detected to explore the role of PxylPBAN in sex pheromone synthesis. 【Methods】 The full-length CDS of PxylPBAN in P. xylostella was cloned using RT-PCR. PxylPBAN was expressed in a prokaryotic expression system and subsequently purified. RNAi was conducted by injecting dsPxylPBAN into female pupae of P. xylostella, followed by the RT-qPCR verification of the expression levels of PxylPBAN, juvenile hormone pathway genes PxylJHAMT, PxylnJHBP, PxylhJHBP and PxylMet, as well as the sex pheromone synthesis pathway genes PxylACC and PxylFAR6 at 24, 48 and 72 h post injection. 【Results】 The cloning process successfully generated a 582 bp full-length CDS for PxylPBAN (GenBank accession number: LOC105391112), of P. xylostella, encoding a 193 amino acid protein with the estimated molecular weight of approximately 21.85 kD. The recombinant PxylPBAN protein was produced through prokaryotic expression. RNAi result revealed a significant down-regulation in expression level of PxylPBAN following the injection of dsPxylPBAN compared to the control group injected with dsEGFP of P. xylostella, with the most pronounced decrease observed at 24 h post injection. Additionally, the expression levels of PxylJHAMT, PxylnJHBP, PxylhJHBP, PxylMet, PxylACC and PxylFAR6 of P. xylostella in the dsPxylPBAN-injected group were significantly reduced as compared to those in the control group. 【Conclusion】 This study successfully obtained the recombinant PxylPBAN protein and identified PxylPBAN as a key gene in the juvenile hormone pathway and sex pheromone synthesis pathway in P. xylostella. These findings establish a theoretical basis for understanding the regulatory relationship between PxylPBAN and juvenile hormones in sex pheromone biosynthesis in P. xylostella.

Key words: Plutella xylostella, pheromone biosynthesis activating neuropeptide, juvenile hormone, gene cloning, prokaryotic expression, RNA interference

郑月琴, 祝潇雯, 田厚军, 林硕, 刘倩霞, 魏辉. 小菜蛾PxylPBAN的克隆、原核表达及其RNAi对保幼激素和性信息素通路基因表达的影响[J]. 昆虫学报, 2025, 68(5): 576-584.

ZHENG Yue-Qin, ZHU Xiao-Wen, TIAN Hou-Jun, LIN Shuo, LIU Qian-Xia, WEI Hui. Cloning and prokaryotic expression of PxylPBAN and its RNAi effects on the expression of genes in the juvenile hormone and sex pheromone pathways in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)[J]. Acta Entomologica Sinica, 2025, 68(5): 576-584.

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小菜蛾PxylPBAN的克隆、原核表达及其RNAi对保幼激素和性信息素通路基因表达的影响

Cloning and prokaryotic expression of PxylPBAN and its RNAi effects on the expression of genes in the juvenile hormone and sex pheromone pathways in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

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