德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

›› 2015, Vol. 58 ›› Issue (10): 1046-1053.

德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

陈静^1,*, 张道伟²

(1. 遵义医学院基础医学院生物化学与分子生物学实验室, 贵州遵义 563000;2. 遵义师范学院生命科学学院, 赤水河流域动物资源保护与应用研究重点实验室, 贵州遵义 563002)

出版日期:2015-10-20 发布日期:2015-10-20
作者简介:陈静, 女, 1982年12月生, 贵州遵义人, 博士, 副教授, 研究方向为昆虫生物化学与分子生物学, E-mail: chenjing_1983@126.com

Molecular cloning, tissue distribution and temperature-induced expression of two trehalose-6-phosphate synthase genes in Blattella germanica (Blattodea: Blattellidae)

CHEN Jing^1,*, ZHANG Dao-Wei²

(1. Laboratory of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Zunyi Medical University, Zunyi, Guizhou 563000, China; 2. Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin, School of Life Sciences, Zunyi Normal College, Zunyi, Guizhou 563002, China)

Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： 【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 Blattella germanica TPS基因，研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列，利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因，分别命名为 BgTPS1 (GenBank登录号：KR050213) 和 BgTPS2 (GenBank登录号：KR050214)。其中，BgTPS1基因cDNA序列全长2 987 bp，开放阅读框 (ORF) 2 502 bp，编码833个氨基酸；BgTPS2基因cDNA序列全长3 212 bp，开放阅读框2 469 bp，编码822个氨基酸。BgTPS1和BgTPS2基因都主要在5龄幼虫脂肪体中表达，且BgTPS2基因的表达量为BgTPS1基因表达量的3.9倍。在两种不同极端温度诱导下，BgTPS1和BgTPS2基因mRNA均上调表达。其中，BgTPS2 的表达量始终显著高于 BgTPS1。在0℃时，BgTPS1和BgTPS2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达，且BgTPS2基因的表达量显著高于BgTPS1基因；低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。

关键词: 德国小蠊, 海藻糖合成酶, 基因克隆, 温度诱导, mRNA表达水平

Abstract: 【Aim】 Trehalose-6-phosphate synthase (TPS) is one of the main enzymes in regulating trehalose levels in the insect haemolymph. This study aims to clone and characterize the TPS genes in Blattella germanica and to profile their expression patterns in extreme temperature environments. 【Methods】 The full-length cDNA sequences of two TPS genes were cloned using RACE technology, and their relative expression levels in different tissues of the 5th instar larvae and under cold (exposed to 10℃ and 0℃ for 1 h, respectively) and heat stress (exposed to 40℃ and 46℃ for 30 min, respectively) were measured using the real-time quantitative PCR (qRT-PCR) technique. 【Results】 The full-length cDNAs oftwo TPS genes were cloned from B. germanica, and named BgTPS1 and BgTPS2, respectively. BgTPS1 (GenBank accession no.: KR050213) is 2 987 bp in length and has an open reading frame (ORF) of 2 502 bp, encoding a protein of 833 amino acids. BgTPS2 (GenBank accession no.: KR050214) is 3 212 bp in length and has an ORF of 2 469 bp, encoding a protein of 822 amino acids. The qRT-PCR analysis revealed that BgTPS1 and BgTPS2 mRNA were mainly expressed in the fat body of the 5th instar larvae, with the level of BgTPS2 mRNA 3.9-fold as high as that of BgTPS1. Under the extreme temperature induction, the expression levels of BgTPS1 and BgTPS2 increased, and the level of BgTPS2 mRNA was much higher than that of BgTPS1 under all stress conditions. The expression levels of both TPS genes were much higher under cold induction (0℃) than under other temperature induction. 【Conclusion】 The two TPS genes were highly expressed in the fat body of the 5th instar larvae of B. germanica, and the level of BgTPS2 mRNA was much higher than that of BgTPS1. The expression levels of BgTPS1 and BgTPS2 increased under both cold and heat stresses. The results provide a foundation for the further research of TPS genes and their roles in the regulation of temperature acclimation in insects.

Key words: Blattella germanica, trehalose-6-phosphate synthase, gene cloning, temperature induction, mRNA expression level

陈静, 张道伟. 德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析[J]. , 2015, 58(10): 1046-1053.

CHEN Jing, ZHANG Dao-Wei. Molecular cloning, tissue distribution and temperature-induced expression of two trehalose-6-phosphate synthase genes in Blattella germanica (Blattodea: Blattellidae)[J]. , 2015, 58(10): 1046-1053.

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