高温胁迫对近江牡蛎Hsp90表达及CpG甲基化的影响

Influence of High Temperature Stress on Hsp90 Expression and CpG Methylation in Crassostrea rivularis

关键词:  高温胁迫  近江牡蛎  热休克蛋白90  核心启动子  甲基化

Key words:  high temperature stress  Crassostrea rivularis  heat shock protein 90  core promoter  methylation

摘要:

为了丰富广温性潮间带底栖生物的温度调控机制研究，本研究采用荧光定量PCR法、亚硫酸氢盐处理基因组DNA结合甲基化特异性PCR（Methylation specific PCR，MSP）法及染色质免疫共沉淀法，对40℃热胁迫24 h后，近江牡蛎（Crassostrea rivularis）热休克蛋白90（Heat shock protein 90，Hsp90）的mRNA表达及其核心启动子甲基化的调控模式进行研究。结果显示：近江牡蛎成贝在40℃热胁迫24 h后，其消化腺、腮和心脏组织中Hsp90 mRNA表达显著升高（P<0.05），核心启动子区CpG平均甲基化水平显著降低（P<0.05），其中邻近热休克元件（Heat shock element，HSE）的CpG2、CpG3、CpG4位点甲基化水平显著降低（P<0.05），CpG5位点甲基化水平无显著变化（P>0.05），灰度分析显示消化腺和心脏组织中的Hsp90核心启动子与RNA聚合酶Ⅱ（RNA polymeraseⅡ）结合增强，腮组织中的Hsp90核心启动子与RNA polymeraseⅡ出现弱结合。表明Hsp90是近江牡蛎抵御高温胁迫的主要表达基因。

Abstract:

In order to enrich the temperature regulation mechanism of benthic organisms in the eurythermic intertidal zone, using methods of qPCR, bisulfite treated genomic DNA combined with methylation specific PCR (MSP) and chromatin immunoprecipitation, the influence of high temperature stress on Hsp90 expression and CpG methylation in Crassostrea rivularis were studied after 24 h heat stress at 40℃. Results showed that after 24 h of heat shock at 40℃,compared to the control group, the expression level of Hsp90 was significantly increased (P<0.05),the average methylation level of core promoter region CpG was significantly decreased (P<0.05),especially the methylation levels of CpG2,CpG3 and CpG4 sites were significantly decreased (P<0.05),which were adjacent to heat shock element (HSE),there was no significant change at methylation level of CpG5 site (P>0.05). Grayscale analysis showed that the binding of Hsp90 core promoter to RNA polymerase Ⅱ was enhanced in digestive gland and heart tissues, and the Hsp90 core promoter in the sputum tissue was weakly bound to RNA polymerase Ⅱ.It indicates that Hsp90 is the main expression gene of C.rivularis against high temperature stress.