柞蚕蛹滞育和滞育解除过程中海藻糖酶基因表达模式及酶活力分析

Gene expression patterns and activities of trehalases in Antheraea pernyi( Lepidoptera: Saturniidae ) pupae during diapause and diapause termination

【目的】克隆柞蚕Antheraea pernyi海藻糖酶(trehalase,Treh)基因,探讨该基因在柞蚕蛹滞育和滞育解除过程中的表达模式与海藻糖酶活力变化,为阐明柞蚕蛹滞育期间糖代谢机制提供参考。【方法】利用RT-PCR技术从柞蚕蛹中克隆获得海藻糖酶基因,并对其进行生物信息学分析。采用半定量RT-PCR检测长光照(17L∶7D)处理后的滞育解除柞蚕蛹与对照滞育蛹不同组织中该基因的表达谱;采用实时定量PCR(qPCR)分析其在长光照下滞育解除过程中柞蚕蛹脂肪体中的相对表达量变化。利用3,5-二硝基水杨酸法检测脂肪体中海藻糖酶活力的变化,同时采用蒽酮比色法测定其血淋巴中海藻糖含量。【结果】克隆获得柞蚕3个海藻糖酶基因,分别命名为ApTreh1A,ApTreh1B和ApTreh2(GenBank登录号分别为:KU977455,KU977456和KU977457),开放阅读框(ORF)全长分别为1 797,1 635和1 932 bp,分别编码598,544和643个氨基酸。同源序列比对与系统进化树分析表明,ApTreh1A和ApTreh1B为可溶型海藻糖酶(Treh S),ApTreh2为膜结合型海藻糖酶(Treh M)。半定量RT-PCR检测发现,各组织中ApTreh2比ApTreh1的分布更广且表达量更高。qPCR检测发现,ApTreh1A和ApTreh1B在长光照处理后的柞蚕蛹脂肪体中,21 d时表达量都表现出快速升高[分别是对照组(12L∶12D)的2倍和4.7倍],28 d与35 d时下降,42 d时表达量再次升高;ApTreh2随着滞育的解除表达量逐渐升高,28 d时达到最高(约为对照组的2.7倍),42 d时又出现一个小高峰(约2.3倍),后期逐渐下降。长光照下脂肪体中海藻糖酶活力逐渐升高,21 d时达到最高(约18.5 U),35 d时降到最低(约11.2 U),42 d时其酶活力再次略微升高,之后呈下降趋势,与基因表达变化趋势一致。蛹血淋巴中海藻糖含量在长光照条件下呈现出升高趋势,21 d时达到最高,在整个发育时期的含量比对照组要高。【结论】本研究结果表明柞蚕蛹滞育解除过程中海藻糖酶基因表达的变化与蛹脂肪体中海藻糖酶活性、蛹血淋巴中海藻糖含量的变化趋势呈一致性,提示海藻糖酶基因的表达响应在柞蚕蛹滞育解除中发挥重要作用。

【Aim】Gene expression patterns and enzyme activities of trehalases in Antheraea pernyi pupae during diapause and diapause termination were detected so as to elucidate the relationship between carbohydrate metabolism and diapause termination during diapause of this insect.【Methods】The trehalase genes were cloned from A.pernyi pupa using RT-PCR technology and subjected to bioinformatics analysis.Their expression patterns in different tissues of A.pernyi pupae during diapause termination after exposure to long photoperiod(17L∶7D)and diapause(the control group)were analyzed by semi-quantitative RT-PCR.The relative expression levels of trehalase genes in the fat body of A.pernyi pupae during diapause termination under long photoperiod were measured using qPCR,the trehalase activity in the fat body was detected using 3,5-dinitrosalicylic acid method and the trehalose content in the haemolymph was measured using antrone chromametry method.【Results】Three trehalase genes were cloned from A.pernyi,named ApTreh1A,ApTreh1B and ApTreh 2 and deposited in GenBank under accession numbers KU977455,KU977456 and KU977457,respectively.Their open reading frames(ORFs)are 1 797,1 635 and 1 932 bp in length,encoding 598,544 and 643 amino acids,respectively.Homologous sequence alignment and phylogenetic analysis indicated that ApTreh1A and ApTreh1B are soluble trehalases(TrehS),and ApTreh2 is a membrane-bound trehalase(TrehM).Tissue-specific mRNA expression profiling using semi-quantitative RT-PCR showed that ApTreh 2 had a wider distribution and higher expression level than ApTreh 1.The qPCR results indicated that the expression levels of ApTreh1A and ApTreh1B in fat body at 21 d after exposure to long photoperiod were up-regulated and significantly higher than that of the control group(12L∶12D)(2-and 4.7-fold,respectively),while down-regulated at 28 and 35 d,and then up-regulated again at 42 d.The expression levels of ApTreh 2 were up-regulated gradually,and reached the maximum(2.7-fold as high as that of the control group)at 28 d.At 42 d after exposure to long photoperiod,there was another expression peak(2.3-fold as high as that of the control group),and then down-regulated gradually.Trehalase activities in fat body increased gradually,reached the peak(about 18.5 U)at 21 d after exposure to long photoperiod,while declined to the lowest level(11.2 U)at 35 d,and increased slightly at 42 d,showing the same variation trend as the gene expression.The trehalose content in the haemolymph increased under long photoperiod and reached the maximum at 21 d,and was higher than that of the control during the whole developmental stage.【Conclusion】The results suggest that the expression of trehalase genes shows the same variation trend as the trehalase activities in the fat body and the trehalose content in the haemolymph in A.pernyi,suggesting that the expression response of trehalase genes plays a significant role in pupal diapause and diapause termination of A.pernyi.