FoxAl Regulating CYP6B6 Expression Under 2-tridecanone Stress in Helicoverpa armigera

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

Abstract

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

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.

Figures/Tables 5

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

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)

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）

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

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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