硫应激单环刺螠差异表达的初步研究

Preliminary Analysis of Gene Expression Differences in Sulfide Stressed Urechis unicinctus

采用mRNA差异显示技术(DDRT-PCR)研究了100μmol/L硫化物环境与正常海水中耐硫生物单环刺螠mRNA的表达差异,初步筛选出7条体腔液细胞的差异表达基因,其中1个cDNA克隆片段与其它物种的MAP kinase kinase kinase(MAPKKK)有较高同源性。进一步采用RT-PCR技术对其在硫化物刺激前后的表达进行了检测,结果显示该基因在对照和应激后2 h6、h的个体中表达较弱;刺激12 h后表达量增高,并随应激时间增加,呈明显上调趋势。表明单环刺螠MAPK信号通路可被硫化物刺激激活,进而通过该信号通路调节下游生理反应,为进一步研究单环刺耐硫特性的分子机制奠定了基础。

The differential display reverse transcriptase-polymerase chain reaction （DDRT-PCR） technique, a versatile technique for the analysis of gene expression that is based on RT-RCR and denaturing polyacrylamide gel electrophoresis, was used to compare the differences in the mRNA expression between normal control and sulfide （100 μmol/L） stressed Urechis unicinctus. Seven differently expressed genes were screened primarily and a different expressed cDNA fragment was found to be highly homogenous with the MAPKKKs of other organisms. Further, with a semi-quantitative RT-PCR technique, the temporal expression of UuMAPKKK-like in U. unicinctus coelomic fluid cells was measured after stimulated by sulfide. The mRNA transcript of UuMAPKKK-like was low in the control and short time stressed （2 h, 6 h） groups, up-regulated gradually after 12 h stimulation, and then reached its maximum level at 48 h. The results showed that MAP kinase signal pathways can be activated by sulfide stress and then regulate downstream physiological effects, providing a foundation for the research of sulfide-resistance mechanism in U. unicinctus.