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不同温度驯化策略诱导优雅蝈螽海藻糖 合成酶基因表达及血淋巴糖类含量变化
Effects of different temperature regimes on the expression of the trehalose-6-phosphate synthase gene and haemolymph sugar content in Gampsocleis gratiosa
骞蕾阳** 寇晓艳 董泽华 常岩林 周志军***
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DOI:10.7679/j.issn.2095-1353.2017.006
作者单位:河北大学生命科学学院,河北省无脊椎动物系统学与应用重点实验室,保定 071002
中文关键词:TPS基因,海藻糖,总糖,温度,实时荧光定量PCR
英文关键词:TPS gene, trehalose, total sugar, temperature, real-time fluorescent quantitative PCR
中文摘要:

【目的】 研究模拟野外温度骤变和缓慢升降温驯化对优雅蝈螽Gampsocleis gratiosa海藻糖合成酶(Trehalose- 6-phosphate synthase, TPS)基因表达量、血淋巴海藻糖及总糖含量的影响以及者间的相关性【方法】 通过高通量转录组数据筛选,从优雅蝈螽中克隆TPS 基因cDNA全长采取模拟野外温度骤变和缓慢升降温两种温度驯化策略以热电偶温度计测得优雅蝈螽体温与环境温度最为接近的温度值作为最适温度,实时荧光定量PCR测定优雅蝈螽TPS表达硫酸蒽酮法测定血淋巴海藻糖和总糖含量(以25℃最适温度作为对照)。 【结果】 克隆得到的TPS基因命名为GgTPSGenBank登录号:KU578006全长3 225 bp,包含2 430 bp的开放阅读框,5′-非编码区(5′-untranslated region, 5′-UTR153 bp3′-非编码区(3′-untranslated region, 3′-UTR642 bp。共编码809个氨基酸,预测的蛋白分子量(Mw91.35 ku,理论等电点(pI6.28。骤然降温阶段TPS相对表达量呈先升高后降低的变化趋势,在15℃最高,与最适温度25℃差异显著。海藻糖相对含量在0℃显著高于25℃。总糖相对含量在0℃10℃显著高于25℃。骤然升温阶段,TPS相对表达量呈先降低后升高,再降低的变化趋势,40℃达到最高且与25℃差异显著。缓慢降温阶段TPS相对表达量呈先升高后降低的变化趋势,在15℃20℃显著高于25℃;海藻糖含量表现出先降低后升高的变化趋势,0℃显著高于25℃,总糖含量在0℃10℃20℃25℃差异显著。缓慢升温阶段TPS相对表达量先降低后升高,30℃45℃显著低于25℃。海藻糖相对含量在40℃显著低于25℃。总糖相对含量呈先升高后降低的变化趋势,40℃45℃显著高于25℃。不同温度驯化策略比较TPS相对表达量在15℃40℃差异显著;海藻糖相对含量在40℃45℃差异显著;总糖相对含量在10℃20℃差异显著;体温在30℃差异显著。【结论】 温度驯化能够提高昆虫的抗逆能力昆虫体内TPS表达量和海藻糖合成量与其所处环境温度及作用时间密切相关低、高温条件下TPS的表达和海藻糖的积累模式不同。

英文摘要:

[Objectives]  To ascertain the effects of rapid vs gradual change in temperature on trehalose-6-phosphate synthase (TPS) gene expression levels, and trehalose, and total sugar, content, in the haemolymph of Gampsocleis gratiosa. [Methods]  Based on the G. gratiosa transcriptome sequencing data set, the full cDNA of the G. gratiosa TPS gene was cloned. Rapid and gradual change in temperature treatments were applied to G. gratiosa. The body temperature of G. gratiosa was measured with a thermocouple thermometer, and the environmental temperature closest to body temperature was regarded as the optimal temperature. TPS gene expression was detected using real-time fluorescent quantitative PCR. Sulfuric acid-anthrone colorimetry was used to measure trehalose, and total sugar, content (the control was the normal content at 25℃). [Results]  The cDNA was cloned and designated as GgTPS (GenBank accession no. KU578006). The full-length cDNA of GgTPS is 3 225 bp with 5′- and 3′-untranslated regions of 153 bp and 642 bp, respectively. It contains an open reading frame of 2 430 nucleotides encoding a protein of 809 amino acids residues with a predicted molecular mass of approximately 91.35 ku and isoelectric point of 6.28. TPS expression levels first increased, then decreased, peaking at 15℃, which was in marked contrast to that measured at 25℃ during the rapid cooling phase. Trehalose content remained constant level before increasing to a high level at 0℃. Total sugar content increased gradually, reaching a maximal level at 0℃. During the rapid heating stage, TPS had a down-up-down expression profile, peaking at 40℃. During the gradual cooling stage, TPS expression first increased, then decreased, and was highly expressed at 15℃ and 20℃. Trehalose content first decreased, then increased, and was significantly higher at 0℃. Total sugar content was markedly different at 0℃, 10℃ and 20℃ to that measured at 25℃. During gradual heating, TPS expression first declined, then increased, and was significantly lower at 30℃ and 45℃. Trehalose content was significantly lower at 40℃ than at 25℃. Total sugar content first increased, then declined, and was significantly higher at 40℃ and 45℃ than at 25℃. Comparing rapid vs gradual change in temperature, TPS expression differed significantly at 15℃ and 40℃, and trehalose content was markedly different at 40℃ and 45℃. Total sugar content was markedly different at 10℃ and 20℃, and body temperature was also markedly different at 30℃. [Conclusion]  Temperature acclimatization improves the ability of insects to resist stress. The TPS expression levels and trehalose content of insects are closely related to ambient temperature, as well as the duration of temperature acclimatization. Patterns of TPS expression and trehalose accumulation in G. gratiosa kept at low temperature differ from those at high temperature. 

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