2-十三烷酮胁迫下棉铃虫FoxAl调控CYP6B6的表达

doi:10.13560/j.cnki.biotech.bull.1985.2021-1305

摘要/Abstract

摘要：

旨在研究2-十三烷酮胁迫对棉铃虫Helicoverpa armigera叉头框A类似蛋白（forkhead box A-like protein,FoxAl）基因表达水平的影响,及FoxAl蛋白是如何调控解毒酶基因CYP6B6表达,为进一步明确FoxAl参与棉铃虫解毒代谢和生长发育过程提供依据。通过酵母自激活检测FoxAl蛋白的转录激活能力;通过凝胶阻滞检测FoxAl蛋白与CYP6B6启动子的结合能力;通过RNAi沉默棉铃虫5龄幼虫中肠内FoxAl基因,检测不同时间（24,48,72和 96 h）后FoxAl和CYP6B6的表达变化情况;最后通过qPCR检测不同浓度（5,10和20 mg/g）2-十三烷酮处理不同时间（6,12,20,30和48 h）后棉铃虫6龄幼虫中肠内FoxAl和CYP6B6的表达谱,并分析FoxAl和CYP6B6表达谱的相关性。FoxAl蛋白具有激活酵母MEL1报告基因转录的能力,且该蛋白能与CYP6B6启动子中响应植物次生物质应答的核心片段结合。利用dsFoxAl沉默棉铃虫5龄幼虫中肠内FoxAl的表达量后,CYP6B6的表达量也显著降低。2-十三烷酮处理棉铃虫6龄幼虫后,中肠内FoxAl和CYP6B6的表达量变化情况相似,基本都呈抛物线趋势,在短时间12 h内两者的表达量迅速上升后,随胁迫时间的延长两者的表达量逐渐降低;此外,FoxAl和CYP6B6的表达量变化基本呈正相关,甚至在20 mg/g的胁迫浓度以及12 h和48 h的胁迫时间下两者都是高度正相关（r=0.819,P=0.045;r=0.987,P=0.007;r=0.978,P=0.011）。棉铃虫FoxAl蛋白可能是CYP6B6的转录激活因子,在植物次生物质2-十三烷酮短期胁迫下,FoxAl的表达量增加,进一步上调CYP6B6的表达,从而参与棉铃虫对2-十三烷酮的解毒作用。

关键词: 棉铃虫, 叉头框类似蛋白, 细胞色素P450酶, 2-十三烷酮, 转录因子

Abstract:

This study aims to investigate the effect of 2-tridecaneone stress on the gene expression of forkhead box A-like protein（FoxAl）in Helicoverpa armigera,and how FoxAl protein regulates the expression of detoxification enzyme gene CYP6B6,so as to provide a basis for further clarifying the role of FoxAl in detoxification metabolism and growth and development of H. armigera. Firstly,yeast self-activation test was applied to detect the transcriptional activation ability of FoxAl protein,and electrophoretic mobility shift assay was used to detect the binding ability of FoxAl protein to CYP6B6 promoter. Expression changes of FoxAl and CYP6B6 after different times（24,48,72 and 96 h）were detected after silencing FoxAl gene in the midgut of 5th H. armigera instar larva by RNAi. Finally qPCR was used to detect the expression profiles of FoxAl and CYP6B6 in the midgut of different 2-tridecone concentrations（5,10 and 20 mg/g）treated 6th instar larvae of H. armigera at different time post treatment（6,12,20,30 and 48 h）,and their Pearson correlation was analyzed. FoxAl protein had the ability to activate transcription of MEL1 reporter gene in yeast,and it bound to the core fragment of CYP6B6 promoter in response to plant secondary substances. Using dsFoxAl to silence FoxAlexpression,the expression of CYP6B6 also significantly reduced in the midgut of 5th instar larvae of H. armigera. After the 6th instar larvae of H. armigera were treated with 2-tridecanone,the expressions of FoxAl and CYP6B6 in the midgut had similarly changed,basically showing a parabolic trend. The expressions of both rapidly increased within 12 h,and then gradually decreased with the extension of stress time. After with 2-tridecaneone treated,the expressions of FoxAl and CYP6B6 were basically positive correlation. They were highly positive correlation coefficients（r=0.819,P=0.045;r=0.987,P=0.007;r=0.978,P=0.011）even at the stress concentration of 20 mg/g and the stress times of 12 and 48 h. In conclusion,FoxAl protein may be a transcriptional activator of CYP6B6 in H. armigera. Under the short-term stress of plant secondary substance such as 2-tridecone,the expression of FoxAl increased,further up-regulated the expression of CYP6B6,thus participating in the detoxification to 2-tridecone in H. armigera.

Key words: Helicoverpa armigera, forkhead box like protein, cytochrome P450 enzyme, 2-tridecanone, transcriptional factor

魏倩, 刘小宁, 赵洁. 2-十三烷酮胁迫下棉铃虫FoxAl调控CYP6B6的表达[J]. 生物技术通报, 2022, 38(5): 84-92.

WEI Qian, LIU Xiao-ning, ZHAO Jie. FoxAl Regulating CYP6B6 Expression Under 2-tridecanone Stress in Helicoverpa armigera[J]. Biotechnology Bulletin, 2022, 38(5): 84-92.

图/表 5

图1 酵母Y2HGold（pGBKT7-FoxAl）菌株的转录激活功能检测 A：pGBKT7-FoxAl质粒的酶切鉴定（M：DL15000 DNA marker;1：pGBKT7-FoxAl质粒;2：pGBKT7-FoxAl质粒的EcoR I和BamH I酶切产物）;B：Y2HGold（pGBKT7-FoxAl）菌株的菌落PCR鉴定（M：DL2000 DNA marker;-：阴性对照;1和2：两个转化后菌株）;C：Y2HGold（pGBKT7-FoxAl）菌株在X-α-gal诱导下的自激活试验

Fig.1 Transcriptional activation test of transformed yeast Y2HGold（pGBKT7-FoxAl）strain A：Restriction digestion of pGBKT7-FoxAl plasmid（M：DL15000 DNA marker;1：pGBKT7-FoxAl plasmid;2：digestion products of pGBKT7-FoxAl plasmid by EcoRI and BamH I）. B：Colony PCR identification of Y2HGold（pGBKT7-FoxAl）strain（M：DL2000 DNA marker;-：negative control;1 and 2：two transformed strains）. C：Self-activation test of Y2HGold（pGBKT7-FoxAl）strain under the induction of X-α-gal

图2 棉铃虫FoxAl蛋白与CYP6B6启动子HE1片段的结合验证 A：凝胶迁移率检测FoxAl蛋白与HE1片段的相互作用 (泳道1: 1.0 ng HE1探针;泳道2: 1.0 ng HE1探针和1 μg FoxAl蛋白;泳道3: 1.0 ng HE1探针和1.5 μg FoxAl蛋白). B：凝胶阻滞试验证实FoxAl蛋白与HE1片段的结合 (泳道1: 1.0 ng HE1探针; 泳道2: 1.0 ng HE1探针和1 μg FoxAl蛋白; 泳道3: 1.0 ng HE1探针、1 μg FoxAl蛋白和标记探针100倍量的无关PGRP-B片段; 泳道4: 1.0 ng HE1探针、1 μg FoxAl蛋白和100倍量的未标记HE1片段)

Fig. 2 Verification of FoxAl in H. armigera binding to the HE1 fragment of CYP6B6 promotor A: Gel-shift assay of interaction between FoxAl and HE1 fragment (Lane 1: 1.0 ng HE1 probe; Lane 2: 1.0 ng HE1 probe and 1 μg FoxAl protein; Lane 3: 1.0 ng HE1 probe and 1.5 μg FoxAl protein). B: EMSA test of FoxAl combination with HE1 fragment (Lane 1: 1.0 ng HE1 probe; Lane 2: 1.0 ng HE1 probe and 1 μg FoxAl protein; Lane 3: 1.0 ng HE1 probe, 1 μg FoxAl protein and 100-fold labelled probe independent PGRP-B fragment; Lane 4: 1.0 ng HE1 probe, 1 μg FoxAl protein and 100-fold unlabelled HE1 fragment)

图3 FoxAl dsRNA 注射后棉铃虫5龄幼虫中肠内FoxAl和CYP6B6的相对表达量在同一时间下用不同星号表示基因的相对表达量与对照组差异显著（*P<0.05,** P<0.01,*** P<0.001）

Fig. 3 Relative expression of FoxAl and CYP6B6 in the midgut of 5th instar larvae after FoxAl dsRNA injected Different asterisks indicate that the gene relative expression is of significant difference compared to control group under the same time（* P<0.05,** P<0.01,*** P<0.001）

图4 2-TD胁迫后棉铃虫6龄幼虫中肠内FoxAl和CYP6B6的相对表达量

Fig. 4 Relative expression of FoxAl and CYP6B6 in the midgut of the 6th instar larvae after 2-TD treated

表1 FoxAl和CYP6B6表达量的相关性分析

Table 1 Correlation analysis of FoxAl and CYP6B6 expre-ssion

2-TD处理2-TD treatment		r	P
胁迫浓度 Stress concentration/（mg·g^-1）	5	0.532	0.178
	10	0.653	0.116
	20	0.819	0.045
胁迫时间 Stress time/h	6	0.614	0.193
	12	0.987	0.007
	20	-0.326	0.337
	30	0.863	0.069
	48	0.978	0.011

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