热胁迫下八字地老虎hsc70基因在不同组织转录表达差异性分析
Analysis of transcription differences of the heat shock cognate 70 gene in different tissues in response to heat stress in Xestia c-nigrum Linnaeus (Lepidoptera: Noctuidae)
王 玲1** 杨 帅2 朱明贺1 徐忠新1 赵奎军1*** 韩岚岚1***
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作者单位：1. 东北农业大学农学院，哈尔滨 150030；2. 黑龙江省农业科学院植物脱毒苗木研究所，哈尔滨 150086
中文关键词：八字地老虎，热激同源蛋白70，实时荧光定量PCR，Western blotting
英文关键词：Xestia c-nigrum Linnaeus, HSC70, Real-time qPCR, western blotting
中文摘要：
【目的】 为探讨八字地老虎Xestia c-nigrum（Linnaeus）Xe-hsc70基因表达与高温耐受性之间的关系，比较热胁迫下Xe-hsc70基因在不同组织中诱导表达的差异。【方法】 本研究以八字地老虎4龄幼虫为研究对象，采用RT-PCR和RACE技术克隆获得编码热激同源蛋白70（70 ku heat shock cognate，HSC70）的基因（命名为Xe-hsc70）cDNA全序列与基因组DNA序列（Genomic DNA，gDNA），并利用实时荧光定量PCR和Western blotting技术，比较分析不同热胁迫温度及不同热诱导时间下八字地老虎4龄幼虫体内马氏管、中肠、体壁、脂肪体与唾腺5个组织中Xe-hsc70基因在mRNA转录与蛋白表达两水平上相对表达量的变化。【结果】 比较分析克隆得到的Xe-hsc70 基因cDNA全序列和gDNA序列，结果表明Xe-hsc70基因含有8个内含子，最大内含子（561 bp）位于5′端非编码区，并含有一个类似热激应答原件HSE的核心结构序列（gaatatgCaGAAtgTTCcaGaa），其余内含子（长度在86~218 bp之间不等）均在编码区内，本研究首次报道了八字地老虎hsc70内含子的具体数目及位置。组织差异性分析显示：在常温25℃条件下，Xe-HSC70在脂肪体中表达量最高，在唾腺中表达量最低；经热激诱导后中肠、唾腺与体壁组织中Xe-HSC70的表达量与对照（25℃）相比显著上调，随着热激时间的延长，表达量呈现出先升高后恢复至对照水平的变化趋势，脂肪体和马氏管Xe-HSC70表达量与对照相比无明显变化。【结论】 八字地老虎不同组织在应对热胁迫的过程中，抗逆机制具有明显的差异。Xe-HSC70的不断积累是八字地老虎对热胁迫不断适应的一个过程，其组织中Xe-hsc70基因的高表达在八字地老虎抗热胁迫的过程中起着重要作用，并为从分子水平上研究八字地老虎的抗逆机理提供依据。
英文摘要：[Objectives] To investigate the effects of heat on Xe-hsc70 expression in Xestia c-nigrum Linnaeus. [Methods] cDNA and gDNA sequences of the heat shock cognate 70 (70 ku heat shock cognate, HSC70) gene ( hereafter Xe-hsc70) were cloned from fourth instar larvae of X. c-nigrum using the RT-PCR and RACE techniques. The relative expression levels of mRNA and protein in different tissues (malpighian tubules, fat body, midgut, epidermis and salivary glands) under a series temperatures and times were determined by real time quantitative PCR and Western blotting. [Results] The Xe-hsc70 gene contains 8 introns, the longest of which lies at 5′-UTR and contains a HSE-like core sequence (gaatatgCaGAAtgTTCcaGaa). The other introns (with lengths from 86 to 218 bp) are located in the ORF. Xe-HSC70 expression was highest in the fat body and lowest in the salivary glands at 25℃. The relative expression levels of Xe-HSC70 in the midgut, salivary glands and epidermis increased significantly after heat shock, and increased with the duration of heat stress; expression levels first increased and then returned to the control level. There was no significant difference in the mRNA expression levels of Xe-HSC70 in the fat body and malpighian tubules compared with the control. [Conclusion] These findings indicate that different tissues of X. c-nigrum respond differently to heat stress. The high expression of the Xe-hsc70 gene plays an important role in the high temperature tolerance of X. c-nigrum, and provides a foundation for further investigation of thermal stress-avoidance mechanisms of X. c-nigrum at the molecular level