›› 2017, Vol. 60 ›› Issue (12): 1384-1393.doi: 10.16380/j.kcxb.2017.12.004

• 研究论文 • 上一篇    下一篇


路标1, 谭瑶1, 周晓榕1, 邢莉2, 庞保平1,*   

  1. (1. 内蒙古农业大学草原昆虫研究中心, 呼和浩特 010019; 2. 内蒙古科学技术研究院, 呼和浩特 010010)
  • 出版日期:2017-12-20 发布日期:2017-12-20

Molecular cloning of trehalose-6-phosphate synthase gene GdTPS and its response to temperature stress in Galeruca daurica (Coleoptera: Chrysomelidae)

LU Biao1, TAN Yao1, ZHOU Xiao-Rong1, XING Li2, PANG Bao-Ping1,*   

  1. (1. Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010019, China; 2. Inner Mongolia Institute of Science and Technology, Hohhot 010010, China)
  • Online:2017-12-20 Published:2017-12-20

摘要: 【目的】海藻糖合成酶(trealose-6-phosphate synthase, TPS)是海藻糖合成过程中的关键酶之一。本研究旨在克隆沙葱萤叶甲Galeruca daurica海藻糖合成酶基因,分析其对不同温度胁迫的响应,以期进一步揭示沙葱萤叶甲耐温性的分子调控机制。【方法】通过RACE技术克隆沙葱萤叶甲TPS基因的全长cDNA序列,并对该基因进行生物信息学分析;利用实时荧光定量PCR方法检测TPS基因在不同温度下沙葱萤叶甲2龄幼虫中的表达量变化。【结果】克隆获得沙葱萤叶甲TPS基因,将其命名为GdTPS(GenBank登录号: KY460114),该基因全长2 706 bp,开放阅读框(ORF)长2 496 bp,编码831个氨基酸;蛋白预测分子量为94.05 kD,等电点为6.82,无信号肽和跨膜结构,包含3个N糖基化位点。GdTPS有两个保守功能区,与其他昆虫TPS具有较高的同源性,其中与马铃薯甲虫Leptinotarsa decemlineata TPS亲缘关系最近,氨基酸序列一致性为88%。实时荧光定量PCR结果表明,温度低于25℃(对照)时,GdTPS表达量随着温度下降而上升,-10℃时达到最高值;高于25℃时,GdTPS表达量随着温度上升而上升,40℃时达到最高值。【结论】沙葱萤叶甲幼虫通过上调GdTPS的表达来应对高温和低温胁迫,该结果为揭示TPS在昆虫应对温度胁迫过程中作用的分子机制提供了基础。

关键词: 沙葱萤叶甲, 海藻糖合成酶, 基因克隆, 温度胁迫, 实时荧光定量PCR

Abstract: 【Aim】 Trehalose-6-phosphate synthase (TPS) is one of key enzymes in the trehalose synthesis pathway. This study aims to clone the TPS gene from Galeruca daurica and to analyze its expression levels in response to temperature stress so as to investigate the molecular regulation mechanisms of TPS in temperature tolerance in G. daurica. 【Methods】 The full-length cDNA of the TPS gene was cloned from G. daurica by rapid amplification of cDNA ends (RACE), and its sequence was subjected to bioinformatics analysis. The expression profiles of the TPS gene in the 2nd instar larvae of G. daurica at different temperatures were detected by real-time quantitative PCR. 【Results】 A TPS gene was cloned from G. daurica, and named GdTPS (GenBank accession no.: KY460114), which is 2 706 bp in length with an open reading frame (ORF) of 2 496 bp, encoding a protein of 831 amino acids with the predicted molecular weight of 94.05 kD and pI of 6.82. The encoded protein contains three potential N-glycosylation sites, without signal peptide and transmembrane domain. GdTPS has two conserved domains, shares higher amino acid sequence identity with TPSs of other insect species and has the highest amino acid sequence identity (88%) with TPS from Leptinotarsa decemlineata. Real-time quantitative PCR results showed that when the stress temperature was lower than 25℃ (the control), the expression level of GdTPS increased as the temperature decreased, and reached the peak at -10℃. However, when the stress temperature was higher than 25℃, its expression level increased as the temperature increased, and reached the peak at 40℃.【Conclusion】 The expression of GdTPS in G. daurica larvae is up-regulated significantly in response to low and high temperaturestresses. The results provide a foundation for the functional research of TPS in the regulation of temperature stress in insects.

Key words: Galeruca daurica, trehalose-6-phosphate synthase, gene cloning, temperature stress, real-time quantitative PCR