草地贪夜蛾海藻糖合成酶基因的克隆、时空表达谱及对温度胁迫的响应

【目的】本研究旨在通过克隆草地贪夜蛾Spodoptera frugiperda的海藻糖合成酶(trehalose-6-phosphate synthase)基因SfTPS，分析其在草地贪夜蛾不同发育阶段、不同组织中的表达水平及不同温度胁迫时5龄幼虫中的相对表达量，为进一步探究TPS在草地贪夜蛾生长发育及抗逆应激反应中的功能奠定基础。【方法】运用RT-PCR技术克隆草地贪夜蛾SfTPS的全长编码区，并进行生物信息学分析。运用RT-qPCR技术检测SfTPS在草地贪夜蛾不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(体壁、中肠和脂肪体)中和经短期(2, 4和8 h)高(35℃)低温(10℃)胁迫后5龄幼虫中的表达变化。【结果】克隆获得2 571 bp的草地贪夜蛾TPS cDNA序列，命名为SfTPS(GenBank登录号： MT920672)，全长开放阅读框(ORF)长2 481 bp，编码的826个氨基酸具有TPS和TPP两个保守结构域。同源比对和系统进化分析表明，昆虫TPS蛋白具有较高的保守性，SfTPS与斜纹夜蛾S. litura的TPS亲缘关系最近，序列一致性达到99.15%。SfTPS中α-螺旋、β-折叠和无规则卷曲占比分别为38.14%, 12.23%和48.55%；SfTPS的三级结构为同源二聚体。RT-qPCR结果表明，SfTPS在草地贪夜蛾卵期和1-5龄幼虫期低表达，在6龄幼虫期、蛹期和成虫期高表达，且草地贪夜蛾变态前后SfTPS的表达量变化较大。组织分布结果显示，SfTPS在5龄幼虫脂肪体中表达量最高。草地贪夜蛾5龄幼虫经2~8 h低温(10℃)和高温(35℃)胁迫后，SfTPS的相对表达量显著高于对照(25℃)，分别为对照的4.43~9.34和2.50~6.03倍。【结论】SfTPS基因在草地贪夜蛾生长发育过程及抵御高低温度胁迫中可能具有重要作用。

Molecular cloning and temporal-spatial expression profiling of trehalose-6-phosphate synthase gene inSpodoptera frugiperda(Lepidoptera: Noctuidae) and its response to temperature stress

【Aim】 This study aims to clone SfTPS gene from the fall armyworm, Spodoptera frugiperda, and to analyze its relative expression levels in different developmental stages, different tissues and the 5th instar larvae under different temperature stresses, so as to lay the foundation for further exploring the function of TPS in the growth and development of S. frugiperda and its anti-stress response. 【Methods】 The full-length coding region of S. frugiperda SfTPS was cloned by RT-PCR technology and analyzed by bioinformatics. The relative expression levels of SfTPS in different developmental stages (egg, 1st-6th instar larva, pupa, and adult), different tissues of the 5th instar larva (integument, midgut, and fat body), and the 5th instar larvae of S. frugiperda subjected to short-term (2, 4 and 8 h) high temperature (35℃) and low temperature (10℃) stress were detected by RT-qPCR technology. 【Results】 The cDNA sequence of TPS gene of 2 571 bp was cloned from S. frugiperda and named SfTPS (GenBank accession number: MT920672). Its open reading frame (ORF) is 2 481 bp in length, encoding 826 amino acids with two conserved domains (TPS and TPP). Homologous comparison and phylogenetic analysis showed that TPS proteins in insects are highly conservative. SfTPS has the closest relationship with TPS of S. litura, showing 99.15% amino acid sequence identity. The proportions of α-helix, β-sheet and random coil in SfTPS are 38.14%, 12.23%, and 48.55%, respectively. The tertiary structure of SfTPS is homo-dimer. The RT-qPCR results showed thatSfTPS was lowly expressed in the egg and 1st-5th instar larval stages of S. frugiperda, and highly expressed in the 6th instar larval, pupal and adult stages. Moreover, the relative expression level of SfTPS changed greatly before and after the metamorphosis of S. frugiperda. The tissue distribution results showed thatSfTPS had the highest expression level in the fat body of the 5th instar larvae of S. frugiperda. After the 5th instar larvae of S. frugiperda were subjected to low temperature (10℃) and high temperature (35℃) stress for 2-8 h, the relative expression levels of SfTPS were significantly higher than that of the control (25℃), being 4.43-9.34- and 2.50-6.03-fold as high as that of the control, respectively. 【Conclusion】 SfTPS gene might play important roles in the growth and development, and resisting high and low temperature stress in S. frugiperda.