Abstract:The primers were designed based on previous reported sequence of metallothionein (MT) gene. Selected genes were amplified by RT-PCR and cloned into pEASY-Blunt vector to construct recombinant plasmid. Standard curves of target gene (MT) or reference gene (β-actin) were generated using serial dilution of recombinant plasmids cDNA. And the method of SYBR Green I real-time quantitative PCR was established for the detection of MT mRNA transcript level. Moreover, MT gene expression level was detected in Eisenia fetida after 14 and 28d of exposure to 50,500,1000mg/kg Cd. The results of sequence alignment showed that the sequences of cDNA fragments in recombinant plasmid were confirmed to be correct corresponding to previous known genes, which indicated that MT and β-actin cDNA fragments were cloned respectively. The liner correlation coefficient of two standard curves was 0.994 and 0.999, respectively and PCR amplification efficiencies were both close to 100%. Therefore, the real-time PCR detection of MT gene expression was sensitive, specific and reliable. Furthermore, the expression level of MT gene was significantly (P<0.01) up-regulated in a dose-dependent and time-dependent manner. The highest (36.8-fold) up-regulation level was found in E. fetida after 28d of exposed to 1000mg/kg Cd compared to controls. These results suggested that MT may be applied as a molecular biomarker provide early warning signs for the stress of Cd exposue in future.
陈春, 周启星. 蚯蚓金属硫蛋白定量PCR检测方法及其分子诊断[J]. 中国环境科学, 2011, 31(8): 1377-1382.
CHEN Chun, ZHOU Qi-Xing. Quantitative real time PCR method for detection of metallothionein in Eisenia fetida and application of molecular diagnosis in cadmium exposure. CHINA ENVIRONMENTAL SCIENCECE, 2011, 31(8): 1377-1382.