蚯蚓金属硫蛋白定量PCR检测方法及其分子诊断

Quantitative real time PCR method for detection of metallothionein in Eisenia fetida and application of molecular diagnosis in cadmium exposure

根据GenBank中蚯蚓(Eisenia fetida)金属硫蛋白(MT)基因序列设计合成引物,采用RT-PCR扩增出目的基因片段,将目的基因片段插入pEASY-Blunt中制备质粒标准品.通过不同梯度稀释倍数的质粒标准品,建立了目的基因MT与内参基因β-actin的SYBR Green I荧光定量RT-PCR检测方法.通过土壤染毒培养实验,将蚯蚓分别暴露于50,500,1000mg/kg重金属Cd染毒的自然土壤中培养14,28d,研究蚯蚓体内MT mRNA转录水平的表达变化.结果表明,重组质粒测序后显示目的片段序列同源性好,证明所设计的MT、β-actin引物能成功用于蚯蚓MT mRNA的检测.两基因构建的荧光定量标准曲线线性关系分别为0.994,0.999,且PCR扩增效率也皆接近于100%.染毒实验发现,Cd可诱导蚯蚓体内MT mRNA的表达,其表达水平与Cd污染暴露呈现出剂量-效应和时间-效应关系,表明蚯蚓MT基因可作为潜在分子标志物,诊断环境中Cd污染及其暴露水平.

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（P0.01） 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.