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荧光标记的酵母细胞用于环境镉的生物监测研究
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Abstract:
目的：前期研究已构建裂殖酵母SPBC776.03敲除株(SPBC776.03Δ)，而且SPBC776.03基因缺失可致细胞周期有丝分裂染色体分离异常。本研究目的是以SPBC776.03Δ为阳性对照，针对环境镉离子的致突变性建立基于酵母的生物监测体系。方法：Rad22-GFP DNA修复灶(DNA repair foci)检测、人工微型染色体丢失实验、以及实时定量PCR (qPCR)。结果：基于PCR以及酵母同源重组，我们成功构建了DNA损伤相关基因Rad22携带荧光标记的酵母菌株Rad22-GFP，用于指示环境中遗传毒因子。镉暴露下的该指示菌株在细胞周期中出现多个(>2)荧光点，而未受镉胁迫的对照组则无。阳性对照SPBC776.03Δ菌株即使未受镉胁迫，其细胞周期中高频出现多个(>2)荧光点。进一步对镉胁迫是否影响SPBC776.03基因转录进行探讨，qPCR测定发现，与未经镉胁迫的野生型酵母相比，镉胁迫导致SPBC776.03转录水平下调3倍，提示SPBC776.03可能为酵母细胞受重金属离子镉胁迫的响应基因。结论：根据本研究工作，已建立了用于环境镉暴露的生物监测体系——Rad22-GFP酵母株，该生物监测周期短，简便易行。
Objectives: Our previous studies have constructed SPBC776.03Δ strain of Schizosaccharomyces pombe, and shown that deficiency of SPBC776.03 gene may lead to mototic chromosome missegregation during cell cycle. This work aims to establish a biomoniting system based on fission yeast to assess the genotoxity and mutangenecity of environmental cadmium ions. Methods: Rad22-GFP DNA repair foci examination, articifical mini-chromosome loss test, and real-time quantitative PCR. Results: Using PCR-based techniques and homologous recombination, a yeast strain called Rad22-GFP was constructed as an indicator of environmental genotoxic agents, in which the DNA damge associated gene Rad22 was tagged with GFP in its chromosomal locus. Under Cd exposure, this indicator strain displayed multiple (>2) DNA repair foci, whereas none was observed with untreated control. As for the positive control strain SPBC776.03Δ, multiple (>2) DNA repair foci were observed during cell cycle even in the absence of Cd stress. Real-time qPCR analyses have revealed that, SPBC776.03 transcriptional levels were found to downregulate by three-fold when exposed to Cd2+, in contrast to untreated wild-type counterparts, suggesting that SPBC776.03 would be a responsive gene of fission yeast to the Cd stress. Conclusions: Our studies have constructed a biomointoring system of envirommental cadmium exposure based on fluorescence-tagged Rad22 expression, Rad22-GFP, in fission yeast, which has advantages of speediness and convenience in the assessment of Cd toxicity.
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