响应槲皮素诱导的美国白蛾P450基因及CYP6ZB2功能分析

doi:10.3864/j.issn.0578-1752.2023.07.010

摘要/Abstract

摘要：

【目的】明确美国白蛾（Hyphantria cunea）响应槲皮素诱导的差异表达P450基因及系统发育关系，探究美国白蛾P450基因对槲皮素诱导的剂量效应，以及P450基因在美国白蛾对槲皮素的解毒适应中的作用。【方法】基于槲皮素诱导的美国白蛾中肠转录组数据，从中筛选显著差异表达的P450基因，应用MEGA X软件对5个P450 基因及其他近缘物种的P450基因进行系统进化关系分析；通过RT-qPCR技术检测5个P450基因在不同槲皮素浓度（0.5%、1.0%、2.0%和4.0%）处理下的表达模式；克隆CYP6ZB2全长cDNA序列，利用RNA干扰（RNAi）技术探明CYP6ZB2基因沉默对美国白蛾体重及存活率的影响。【结果】从转录组的差异表达基因中筛选到5个显著响应槲皮素诱导的P450基因，经P450命名委员会分别被命名为CYP4G296、CYP4G297、CYP333A30、CYP304F33和CYP6ZB2（GenBank登录号：ON032363—ON032367）。系统发育分析表明，这5个基因被聚类到CYP2、CYP3、CYP4和线粒体CYP进化枝。RT-qPCR结果表明，P450基因响应槲皮素胁迫具有显著的剂量效应，CYP4G297、CYP304F33、CYP333A30和CYP4G296的表达均被不同浓度槲皮素抑制，CYP6ZB2的表达在0.5%—2.0%的槲皮素诱导下显著上调，而在4.0%的槲皮素浓度下被显著抑制。克隆CYP6ZB2的全长cDNA序列，CYP6ZB2具有完整的开放阅读框（ORF），编码500个氨基酸。RNAi结果表明，注射两种剂量（500和1 000 ng）dsCYP6ZB2均可显著抑制CYP6ZB2的表达，注射1 000 ng dsCYP6ZB2 48 h后干扰效率达到最高（96.9%）。CYP6ZB2被干扰的美国白蛾幼虫经0.5%槲皮素处理后体重增长量低于对照组，存活率（53.33%）分别为未处理组的61.5%和注射双蒸水组的72.7%（P<0.05）。【结论】筛选鉴定到美国白蛾响应槲皮素诱导的5个P450基因，槲皮素对5个基因的诱导表达具有明显的剂量效应，说明不同的P450基因对槲皮素的响应模式存在差异。RNAi能高效沉默CYP6ZB2，0.5%槲皮素对CYP6ZB2被干扰的美国白蛾幼虫的生长发育具有一定抑制作用，表明CYP6ZB2在美国白蛾对槲皮素的解毒适应中发挥着重要作用。

关键词: 美国白蛾, 槲皮素, P450基因, 表达分析, RNA干扰, 基因功能

Abstract:

【Objective】 The objective of this study is to clarify the differentially expressed cytochrome P450 genes and phylogenetic relationships in response to quercetin in Hyphantria cunea, and to explore the dosage effect of cytochrome P450 genes in response to quercetin, and the role of cytochrome P450 genes in detoxification adaptation to quercetin in H. cunea.【Method】 Based on the transcriptome data of quercetin-induced midgut of H. cunea, the significantly differentially expressed cytochrome P450 genes were screened. MEGA X software was used to analyze the phylogenetic relationships of the target genes with cytochrome P450 genes of other related species. RT-qPCR was used to detect the expression of the five cytochrome P450 genes in different quercetin concentrations (0.5%, 1.0%, 2.0% and 4.0%). The full-length cDNA sequence of CYP6ZB2 was cloned, and the effect of CYP6ZB2 gene-silenced on the body weight and survival rate of H. cunea was investigated by RNA interference (RNAi) technique.【Result】 Five cytochrome P450 genes were identified from the differentially expressed genes in the transcriptome, and were named CYP4G296, CYP4G297, CYP333A30, CYP304F33 and CYP6ZB2 by Standardized Cytochrome P450 Nomenclature Committee (GenBank accession numbers: ON032363-ON032367). Phylogenetic tree analysis showed that these five genes were divided into four clades (CYP2, CYP3, CYP4 and the mitochondrial CYP clade). RT-qPCR results showed that cytochrome P450 genes had a significant dosage effect in response to quercetin, and the expressions of CYP4G297, CYP304F33, CYP333A30 and CYP4G296 were inhibited by different concentrations of quercetin. The expression of CYP6ZB2 was significantly increased at 0.5%-2.0% concentration of quercetin, but it was significantly inhibited at 4.0% quercetin. The full-length cDNA sequence of CYP6ZB2 was cloned. CYP6ZB2 had a complete open reading frame (ORF) encoding 500 amino acids. RNAi results showed that the expression level of CYP6ZB2 was significantly inhibited by injection of two doses (500 and 1 000 ng) dsCYP6ZB2, and the maximum reduction in CYP6ZB2 expression level was 96.9% after injection of 1 000 ng dsCYP6ZB2 for 48 h. The weight increase of CYP6ZB2 gene-silenced H. cunea larvae fed on 0.5% quercetin was lower than that of the control group. Furthermore, the survival rate (53.33%) was 61.5% of that of the untreated group and 72.7% of that of the ddH₂O group (P<0.05).【Conclusion】 Five cytochrome P450 genes were identified in the transcriptome data of quercetin-induced midgut of H. cunea, and quercetin had a significant dosage effect on the five genes, indicating that the response patterns of different cytochrome P450 genes to quercetin were different. RNAi can significantly inhibit the expression level of CYP6ZB2, and quercetin has a significant negative impact on the growth of H. cunea, suggesting that CYP6ZB2 plays an important role in quercetin detoxification in H. cunea.

Key words: Hyphantria cunea, quercetin, cytochrome P450, expression analysis, RNA interference (RNAi), gene function

邵红扬, 孟香, 张涛, 陈敏. 响应槲皮素诱导的美国白蛾P450基因及CYP6ZB2功能分析[J]. 中国农业科学, 2023, 56(7): 1322-1332.

SHAO HongYang, MENG Xiang, ZHANG Tao, CHEN Min. Analysis of Cytochrome P450 Genes in Response to Quercetin and Function of CYP6ZB2 in Hyphantria cunea[J]. Scientia Agricultura Sinica, 2023, 56(7): 1322-1332.

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基因名称 Gene name	引物序列 Primer sequence	扩增效率 Amplification efficiency (%)	相关系数 Correlation coefficient	引物用途 Primer usage
CYP4G296	F-CGACTCAGATCGCCCAGTAA R-TAACAGGCACTGGTGGGAAC	99.2	0.995	RT-qPCR
CYP4G297	F-ACAACAGTAGTGATTGCCACG R-TCGGGCAGGAAATTATCAGGA	106.4	0.997	RT-qPCR
CYP333A30	F-GGTGGCTATCACATCCCTCC R-TCGCCCGCGGATAATACTTT	91.4	0.984	RT-qPCR
CYP304F33	F-AACTTACGCTCGCCAGACAA R-TGCAGGCTTCAGTAATGGCT	99.9	0.988	RT-qPCR
CYP6ZB2	F-ACCCGAGAAATTTGACCCAGA R-TACACACACGCCGAATTGGA	103.5	0.993	RT-qPCR
GAPDH	F-GACTGGCATGGCTTTCAGAG R-CATCGTAGCTAGCGGGTTTG	96.5	0.999	RT-qPCR
EF1-α	F-TTATCGTCGCTGCTGGTACT R-GAGTGTGAAAGCGAGCAGAG	103.5	0.999	RT-qPCR
CYP6ZB2	F-ATGTCGGCTTTCTTACTGCTGT R-TTAGATTGGTATAAAATTGAGATATATGTCT			基因克隆 Gene cloning
dsCYP6ZB2	F-TAATACGACTCACTATAGGG ATGTCGGCTTTCTTACTGCTGT R-TAATACGACTCACTATAGGGTTAGATTGGTATAAAATTGAGATATATGTCT			dsRNA合成 dsRNA synthesis

基因名称Gene name	登录号 Accession number	转录水平 Transcription level	编码蛋白长度 Encoded protein length (aa)	Best B<BOLD>LASTX</BOLD> match
基因名称Gene name	登录号 Accession number	转录水平 Transcription level	编码蛋白长度 Encoded protein length (aa)	蛋白质名称 Protein name	物种 Species	E值 E-value	一致性 Identity (%)	登录号 Accession number
CYP4G296	ON032363	Down (12%)	362	Cytochrome P450 CYP4G	黑肾卷裙夜蛾 Plecoptera oculata	0	90.11	QFS21543.1
CYP4G297	ON032364	Down (5%)	146	Cytochrome P450 CYP4G	美国白蛾 H. cunea	6e-173	100.00	BBD13397.1
CYP333A30	ON032365	Down (24%)	507	Cytochrome P450 CYP12A2-like	粉纹夜蛾 T. ni	0	63.85	XP_026734554.1
CYP304F33	ON032366	Down (25%)	289	CYP15C1	大豆夜蛾 Anticarsia gemmatalis	3e-179	79.29	UBY12684.1
CYP6ZB2	ON032367	Up (2.69倍 2.69 times)	500	Cytochrome P450 6B6-like	棉铃虫 H. armigera	8e-171	51.65	XP_021201217.1