| 陈凯莉,孙春苗,Arina Nur Faidah,张佳雯,曹传旺,孙丽丽,2026,美国白蛾表皮蛋白基因克隆及对HcNPV胁迫响应[J].环境昆虫学报,(4):1208-1219 |
| 美国白蛾表皮蛋白基因克隆及对HcNPV胁迫响应 |
| Cloning of the cuticular protein genes in Hyphantria cunea and its response to HcNPV stress |
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| DOI：10.3969/j.issn.1674-0858.2026.04.21 |
| 中文关键词: 美国白蛾 表皮蛋白 时空表达 美国白蛾核型多角体病毒 |
| 英文关键词:Hyphantria cunea cuticular protein spatiotemporal expression Hyphantria cunea nucleopolyhedrovirus (HcNPV) |
| 基金项目:国家自然基金面上项目（32171791） |
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| 摘要点击次数: 92 |
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| 中文摘要: |
| 表皮在昆虫生长发育和抵御外界伤害和逆境中起重要作用。【目的】本研究旨在克隆美国白蛾Hyphantria cunea表皮蛋白家族cuticular protein（CPs）基因，明确该家族基因特性和时空表达模式，阐明其对美国白蛾核型多角体病毒（Hyphantria cunea nucleopolyhedrovirus，HcNPV）的敏感性。【方法】通过RT-PCR技术克隆HcCPs基因的全长cDNA序列，通过生物信息学在线网站和软件分析HcCPs基因的生物学特性；使用RT-qPCR技术检测HcCPs基因在美国白蛾不同发育阶段（卵、1~7龄幼虫、蛹和成虫）、不同组织（头、表皮、马氏管、前肠、中肠、后肠、丝腺、脂肪体、精巢和卵巢）中及不同浓度HcNPV胁迫下的表达水平。【结果】美国白蛾7个CP基因的开放阅读框（ORFs）分别长291、492、720、426、657、408和942 bp，依次编码96、163、239、141、218、135和313个氨基酸；蛋白分子量为10.8~32.2 kDa，理论等电点为5.00~9.44；三级蛋白结构预测显示，HcCPs的三级结构含α螺旋、β折叠和无规则卷曲等结构。HcCPs系统发育树分析表明，HcCPs与鳞翅目昆虫的亲缘关系更近。RT-qPCR结果表明，HcCPs在美国白蛾头部、表皮、精巢及5~7龄中的表达水平较高，雄成虫中的表达量高于雌成虫。不同浓度HcNPV胁迫美国白蛾幼虫对表皮蛋白基因HcCPs的表达量具有时间效应，高浓度（2×105 PIBs/mL）和低浓度HcNPV（2×103 PIBs/mL）胁迫下，HcCPs的转录水平呈现先升高再降低后升高的趋势；在2×103 PIBs/mL HcNPV的胁迫下，HcCP1、HcCP19、HcPCP36a和HcLCPA2B在120 h表达量最高，为对照组的38.94~4 994.20倍，HcPCP、HcL/PRCP66和HcLCP30在48 h表达量最高，依次为对照组的152.16倍、512.07倍和88.82倍；而在2×105 PIBs/mL HcNPV的胁迫下，HcCP1在24 h表达量最高，是对照组的104.12倍，HcCP19、HcPCP36a和HcLCPA2B在120 h表达量最高，分别是对照组的67.93倍、6 824.97倍和12.66倍，HcPCP、HcL/PRCP66和HcLCP30在48 h表达量最高，分别是对照组的85.91倍、218.66倍和29.16倍，表明HcCPs基因积极应对HcNPV的感染。【结论】这一系列结果证实了HcCPs在美国白蛾抵御HcNPV侵染过程中发挥重要作用，研究结果为阐明HcNPV对美国白蛾的感染机制奠定了基础。 |
| 英文摘要: |
| The insect cuticle plays critical roles in growth and development and provides protection against external injuries and environmental stresses.【Aim】This study aimed to clone cuticular protein (CP) family genes from the fall webworm, Hyphantria cunea, characterize their sequence features and spatiotemporal expression patterns, and investigate their transcriptional responses to Hyphantria cunea nucleopolyhedrovirus (HcNPV) infection.【Methods】The full-length cDNA sequences of HcCPs genes were cloned using RT-PCR, and their molecular and biological characteristics were analyzed using online bioinformatics tools and software. RT-qPCR was applied to detect the expression profiles of the HcCPs genes across different developmental stages (eggs, 1st–7th instar larvae, pupae and adults), in different tissues (head, cuticle, Malpighian tubules, foregut, midgut, hindgut, silk gland, fat body, testis and ovary), and following exposure to different concentrations of HcNPV.【Results】The open reading frames (ORFs) of the seven H. cunea CP genes were 291, 492, 720, 426, 657, 408 and 742 bp in length, encoding proteins of 96, 163, 239, 141, 218, 135 and 313 amino acids, respectively. Their predicted molecular weights ranged from 10.8 to 32.2 kDa, and theoretical isoelectric points ranged from 5.00 to 9.44. Tertiary structure prediction revealed that HcCP proteins contained mainly of α-helices, β-sheets and random coils. Phylogenetic analysis demonstrated that the HcCPs were most closely related to homologous proteins from other lepidopteran insects. RT-qPCR analysis revealed relatively high expression levels of the HcCP genes in the head, cuticle, testis and during the 5th to 7th larval instars. Their expression levels were also higher in male adults than in female adults. Exposure of H. cunea larvae to different concentrations of HcNPV resulted in time-dependent changes in HcCP expression. Under both high-dose (2 × 10? PIBs/mL) and low-dose (2 × 103 PIBs/mL) treatments, transcriptional levels initially increased, subsequently decreased, and then increased again. Following exposure to 2 × 103 PIBs/mL HcNPV, HcCP1, HcCP19, HcPCP36a and HcLCPA2B reached their highest expression levels at 120 h, ranging from 38.94–4 994.20-fold relative to the control. In contrast, HcPCP, HcL/PRCP66 and HcLCP30 peaked at 48 h, with expression levels 152.16-, 512.07- and 88.82-fold higher than those of the control, respectively. Following exposure to 2 × 10? PIBs/mL HcNPV, HcCP1 achieved its highest expression level at 24 h (104.12-fold of control). The expression levels of HcCP19, HcPCP36a and HcLCPA2B peaked at 120 h and were 67.93-, 6 824.97- and 12.66-fold higher than those of the control, respectively. Meanwhile, HcPCP, HcL/PRCP66 and HcLCP30 reached their highest expression levels at 48 h, at 85.91-, 218.66- and 29.16-fold those of controls, respectively. These findings indicate that HcCP genes are strongly involved in the response of H. cunea to HcNPV infection.【Conclusion】Collectively, these results demonstrate that HcCPs play vital roles in the defense of H. cunea against HcNPV infection and provide a foundation for further elucidating the mechanisms underlying HcNPV infection in this insect. |
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