| 陈凯莉，孙春苗，Arina Nur Faidah，张佳雯，曹传旺，孙丽丽,,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,(): |
| 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： |
| 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: |
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| 摘要点击次数: 4 |
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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。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。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。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742 bp，依次编码96、163、239、141、218、135和313个氨基酸；蛋白分子量为1.08~3.22 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。同时，这一系列结果也证实了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, development, and defense against environmental stressors. This study aims to clone the cuticular protein (CP) genes of Hyphantria cunea and investigate their molecular characteristics, spatiotemporal expression patterns, and responses to Hyphantria cunea nucleopolyhedrovirus (HcNPV) infection. Seven HcCP genes were cloned using RT-PCR, and their biological features were analyzed through bioinformatics tools. RT-PCR was used to profile the HcCP expression across the developmental stages (egg, 1st ~ 7th instar larva, pupa, and adult), tissues (head, cuticle, malpighian tubule, foregut, midgut, hindgut, silk gland, fat body, testis, and ovary) and under HcNPV challenges (2×103 and 2×10? PIBs/mL). The open reading frames (ORFs) of seven CP genes ranged from 291 to 742 bp, encoding polypeptides of 96–313 amino acids with predicted molecular weight of 1.08 ~ 3.22 kD and the theoretical isoelectric point of 5.00 ~ 9.44. The tertiary protein structure prediction revealed conserved α-helices, β-sheets, and random coils. Phylogenetic analysis demonstrated close evolutionary relationships with lepidopteran orthologs. Spatiotemporal expression profiling showed predominant HcCP expression in the head, cuticle, and testis, with higher levels in 5th–7th instar larvae and male adults compared to females. The expression of HcCPs in H. cunea larvae exposed to varying concentrations of HcNPV exhibited a time-dependent effect. Under the HcNPV treatment of high concentration (2×105 PIBs/mL) and low concentration (2×103 PIBs/mL), the transcription levels of HcCPs displayed a biphasic pattern characterized by an initial increase, followed by decrease, and subsequently a secondary upregulation. When exposed to 2×103 PIBs/mL HcNPV, HcCP1 ~ 4 exhibited peak expression at 120 h, with fold-changes ranging from 38.94 ~ 4 994.20 compared to the control, while HcCP5 ~ 7 reached maximum transcriptional levels at 48 h , showing 152.16, 512.07, and 88.82 fold increases, respectively. Under 2×105 PIBs/mL HcNPV stress, HcCP1 demonstrated maximal upregulation (104.12-fold) at 24 h, whereas HcCP2 ~ 4 peaked at 120 h with 67.93-, 6,824.97-, and 12.66-fold increases, respectively. Concurrently, HcCP5 ~ 7 showed peak expression at 48h, with 85.91, 218.66, and 29.16 fold increases compared to the control, respectively. These results demonstrate that HcCPs are dynamically regulated during HcNPV infection, suggesting their active involvement in antiviral defense mechanisms. This study provides molecular insights into the interactions between H. cunea CPs and HcNPV, laying a foundation for understanding viral pathogenesis and developing targeted biocontrol strategies. |
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