单 位：
遵义医学院基础医学院生物化学与分子生物学实验室;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
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
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Bg TPS1(Gen Bank登录号:KR050213)和Bg TPS2(Gen Bank登录号:KR050214)。其中,Bg TPS1基因c DNA序列全长2 987 bp,开放阅读框(ORF)2 502 bp,编码833个氨基酸;Bg TPS2基因c DNA序列全长3 212 bp,开放阅读框2 469 bp,编码822个氨基酸。Bg TPS1和Bg TPS2基因都主要在5龄幼虫脂肪体中表达,且Bg TPS2基因的表达量为Bg TPS1基因表达量的3.9倍。在两种不同极端温度诱导下,Bg TPS1和Bg TPS2基因mRNA均上调表达。其中,Bg TPS2的表达量始终显著高于Bg TPS1。在0℃时,Bg TPS1和Bg TPS2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Bg TPS2基因的表达量显著高于Bg TPS1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。
译 名：
Molecular cloning, tissue distribution and temperature-induced expression of two trehalose-6-phosphate synthase genes in Blattella germanica( Blattodea: Blattellidae)
作 者：
CHEN Jing;ZHANG Dao-Wei;Laboratory of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Zunyi Medical University;Key Laboratory of Protection and Utilization of Animal Resource in Chishui River Basin,School of Life Sciences,Zunyi Normal College;
关键词：
Blattella germanica;;trehalose-6-phosphate synthase;;gene cloning;;temperature induction;;mRNA expression level
摘 要：
【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 c DNA 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 c DNAs of two TPS genes were cloned from B. germanica,and named Bg TPS1 and Bg TPS2,respectively.Bg TPS1( Gen Bank 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. Bg TPS2( Gen Bank 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 Bg TPS1 and Bg TPS2 mRNA were mainly expressed in the fat body of the 5th instar larvae,with the level of Bg TPS2 mRNA 3. 9-fold as high as that of Bg TPS1. Under the extreme temperature induction,the expression levels of Bg TPS1 and Bg TPS2 increased,and the level of Bg TPS2 mRNA was much higher than that of Bg TPS1 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 Bg TPS2 mRNA was much higher than that of Bg TPS1. The expression levels of Bg TPS1 and Bg TPS2 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.
