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

作　　者：骞蕾阳 寇晓艳[1] 董泽华[1] 常岩林[1] 周志军[1] 

机构地区：[1]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

出　　处：《应用昆虫学报》2017年第1期56-67,共12页Chinese Journal of Applied Entomology

基　　金：国家自然科学基金面上项目(31471985)

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三者间的相关性。[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GgTPS(GenBank登录号:KU578006),全长3 225bp,包含2 430bp的开放阅读框,5'-非编码区(5'-untranslated region,5'-UTR)153 bp和3'-非编码区(3'-untranslated region,3'-UTR)642 bp。共编码809个氨基酸,预测的蛋白分子量(Mw)91.35 ku,理论等电点(pI)6.28。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℃和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℃和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℃和20℃与25℃差异显著。缓慢升温阶段TPS相对表达量先降低后升高,30℃和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℃和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℃和45℃差异显著;总糖相对含量在10℃和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�[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 eDNA was cloned and designated as GgTPS （GenBank accession no. KU578006）. The full-length eDNA 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

关 键 词：TPS基因 海藻糖 总糖 温度 实时荧光定量PCR 

分 类 号：Q966[生物学—昆虫学]