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| 草地贪夜蛾半胱氨酸蛋白酶Sf-caspase-3基因克隆与表达分析 |
| Cloning and expression analysis of caspase-3 in Spodoptera frugiperda |
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| DOI： |
| 中文关键词: 草地贪夜蛾 Sf-caspase-3 克隆 时空表达 原核表达 |
| 英文关键词:Spodoptera frugiperda Sf-caspase-3 spatio-temporal expression prokaryotic expression |
| 基金项目:广东省重点领域研发计划项目（2020B020223004）；广东省教育厅创新团队项目（2017KCXTD018）；广州市亚热带果树重大疫情控制重点实验室项目（201805010008） |
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| 中文摘要: |
| 半胱氨酸蛋白酶caspases是细胞凋亡过程中重要的酶类，参与调控昆虫变态发育及响应外界压力等多种生理过程。本研究通过转录组数据及逆转录PCR鉴定并验证得到Sf-caspase-3基因编码区全长。该基因编码区长840 bp，编码279个氨基酸，预测蛋白分子量为31.43 kDa。氨基酸序列分析表明Sf-caspase-3具有保守的QACRG五肽序列，且与Lep-caspase-3高度保守。进化树分析表明Sf-caspase-3与甜菜夜蛾Spodoptera exigua Se-caspase-3亲缘关系最近。RT-qPCR检测结果表明Sf-caspase-3在草地贪夜蛾Spodoptera frugiperda各个龄期均有表达，其中3~6龄幼虫期表达量最高，蛹期和卵期表达量最低。此外Sf-caspase-3在6龄幼虫中肠组织中表达量最高，而在表皮、脂肪体及马氏管中表达量极低。本研究成功构建Sf-caspase-3原核表达重组质粒，在28℃下0.4 mmol/L异丙基硫代半乳糖苷（IPTG）诱导重组菌株4 h得到约50 kDa大小条带，与预期条带大小相符。蛋白可溶性分析表明Sf-caspase-3主要以不可溶的形式存在包涵体中。本研究为草地贪夜蛾Sf-caspase-3功能及鳞翅目昆虫细胞凋亡机制研究提供一定的理论依据。 |
| 英文摘要: |
| Cysteine ??proteases caspases are important enzymes in the process of apoptosis and participate in various insect physiological processes such as regulating the metamorphosis and responding to external pressure. In this study, the full length of the coding region of Spodoptera frugiperda Sf-caspase-3 gene was identified and verified by transcriptome data and reverse transcription PCR, respectively. The gene coding region is 840 bp long and encodes 279 amino acids, with a predicted protein molecular weight of 31.43 kDa. Amino acid sequence analysis showed that Sf-caspase-3 has a conserved QACRG pentapeptide sequence and is highly conserved with Lep-caspase-3. Phylogenetic tree analysis showed that Sf-caspase-3 had the closest relationship with Spodoptera exigua Se-caspase-3. RT-qPCR results showed that Sf-caspase-3 was expressed at every developmental stage of S. frugiperda, among which the 3~6th instar larval stages owned the highest epression, and pupal and egg stages showed with the lowest expression. For different tissues of 6th instar larvae, the expression level of Sf-caspase-3 was the highest in the midgut, but was extremely low in the cuticle, fat body, and malpighian tubules. Besides, Sf-caspase-3 prokaryotic expression recombinant plasmid was successfully constructed. A band of about 50 kDa was identified by SDS-PAGE of the recombinant strain sample which induced by 0.4 mmol/L isopropylthiogalactoside (IPTG) at 28℃ for 4 h. Protein solubility analysis showed that Sf-caspase-3 mainly existed as insoluble form in inclusion bodies. This study provides a theoretical basis for the function of Sf-caspase-3 and the mechanism of lepidopteran insect apoptosis. |
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