Sli-miR-34-5p响应植物次生物质正调控斜纹夜蛾谷胱甘肽S-转移酶基因SlGSTe1的表达

doi:10.16380/j.kcxb.2019.01.001

摘要/Abstract

摘要：

【目的】昆虫解毒酶系统是昆虫适应植物次生物质的主要机制，研究调控解毒酶基因表达的miRNA将发现可用于害虫防治的靶标分子。本研究旨在探究Sli-miR-34-5p对斜纹夜蛾Spodoptera itura中应对植物次生物质的解毒酶谷胱甘肽S-转移酶(GST)基因的调控作用。【方法】通过实时荧光定量PCR检测Sli-miR-34-5p在斜纹夜蛾5龄第1天幼虫进食芥菜Brassica juncea后中肠中的表达；在斜纹夜蛾5龄第1天幼虫体内及细胞株中超表达Sli-miR-34-5p模拟体类似物(mimics)或抑制剂(inhibitors)，采用实时荧光定量PCR和Western blot分析Sli-miR-34-5p已知或推测的靶基因的表达变化；利用双荧光素酶报告系统验证Sli-miR-34-5p对靶标基因的表达调控作用；采用生物信息学方法预测Sli-miR-34-5p可能的靶基因，以了解其可能的作用机理。【结果】结果表明，Sli-miR-34-5p在斜纹夜蛾5龄第1天幼虫进食芥菜24和48 h时的中肠中表达显著上调。在斜纹夜蛾5龄第1天幼虫血淋巴中

超表达Sli-miR-34-5p mimic，进食芥菜的斜纹夜蛾中SlGSTe1转录上调；反之，超表达Sli-miR-34-5p inhibitor，SlGSTE1蛋白水平显著下调。在斜纹夜蛾Spli-221细胞株中添加芥菜次生物质吲哚-3-甲醇(I3C)，Sli-miR-34-5p和SlGSTe1的表达量均呈现上调的趋势；此外，在细胞中转染Sli-miR-34-5p mimic，SlGSTe1的表达水平明显上调。然而，生物信息学预测和双荧光素酶报告系统检测结果显示，Sli-miR-34-5p并不直接作用于靶基因SlGSTe1。【结论】研究结果提示Sli-miR-34-5p通过促进斜纹夜蛾谷胱甘肽S-转移酶基因SlGSTe1的表达而参与斜纹夜蛾应对植物次生物质的胁迫。

关键词: 斜纹夜蛾, miRNA, Sli-miR-34-5p, 解毒酶, 表达调控, 植物次生物质

Abstract: 【Aim】 Insect detoxification enzymes play important roles in insect adaptation to secondary plant substances. Studying the miRNAs, which regulate the expression of detoxification enzyme genes, will help to identify the target for pest control. This study aims to investigate the regulation of Sli-miR-34-5p on the detoxification enzyme glutathione S-transferase (GST) gene involved in the adaptation to secondary plant substances in Spodoptera litura. 【Methods】 The expression level of Sli-miR-34-5p in the midgut of the day-1 5th instar larvae of S. litura fed with Brassica juncea was determined by qPCR. After the overexpression of Sli-miR-34-5p mimic or inhibitor, the expression of the known or presumable target gene of Sli-miR-34-5p at the mRNA and protein levels in S. litura larvae was determined by qPCR and Western blot, respectively. The regulation of Sli-miR-34-5p on its target gene was verified by dual luciferase reporter system. The possible action mechanism of Sli-miR-34-5p on target genes was investigated by predicting the target genes of Sli-miR-34-5p with bioinformatics method.【Results】 The results showed that the expression of Sli-miR-34-5p was up-regulated in the midgut of the day-1 5th instar larvae of S. litura fed with B. juncea for 24 and 48 h. The injection of Sli-miR-34-5p mimic into the hemolymph of the day-1 5th instar larvae of S. litura resulted in the up-regulation of the mRNA level of SlGSTe1 in the midgut of B. juncea-fed larvae. Injection of Sli-miR-34-5p inhibitor into the hemolymph significantly downregulated the protein level of SlGSTE1. In the S. litura Spli-221 cell line treated with indole-3-carbinol, a secondary substance of B. juncea, the expression levels of both Sli-miR-34-5p pression of SlGSTe1 enhanced after Sli-miR-34-5p mimic was added to the cell line. However, the assay of dual luciferase activity showed that Sli-miR-34-5p did not directly bind to SlGSTe1Sli-miR-34-5p nformatics analysis showed that SlGSTe1 may be not the direct target gene of Sli-miR-34-5p. 【Conclusion】 The results suggest that Sli-miR-34-5p may be involved in the response of S. litura to secondary substances of host plants by enhancing the expression of the glutathione S-transferase gene SlGSTe1.

Key words: Spodoptera litura; miRNA, Sli-miR-34-5p, detoxification enzyme, expression regulation, secondary plant substances

马康, 邹晓鹏, 岑永杰, 郑思春. Sli-miR-34-5p响应植物次生物质正调控斜纹夜蛾谷胱甘肽S-转移酶基因SlGSTe1的表达[J]. 昆虫学报, 2019, 62(1): 1-8.

MA Kang, ZOU Xiao-Peng, CEN Yong-Jie, ZHENG Si-Chun. Sli-miR-34-5p positively regulates the expression of the glutathione S-transferase gene SlGSTe1 in Spodoptera litura (Lepidoptera: Noctuidae) in response to secondary plant substances[J]. Acta Entomologica Sinica, 2019, 62(1): 1-8.

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Sli-miR-34-5p响应植物次生物质正调控斜纹夜蛾谷胱甘肽S-转移酶基因SlGSTe1的表达

Sli-miR-34-5p positively regulates the expression of the glutathione S-transferase gene SlGSTe1 in Spodoptera litura (Lepidoptera: Noctuidae) in response to secondary plant substances

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