A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.
A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.
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Fox1 在莱茵衣藻中的表达需要 C2H2 锌指蛋白 FEMU2。
DOI：
10.1371/journal.pone.0112977
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Fei X
中科院分区：
文献类型：
--
作者：
Deng X;Yang J;Wu X;Li Y;Fei X
莱茵衣藻fox 1基因编码一种铁氧化酶，该酶参与细胞铁的吸收，在缺铁条件下高度诱导。为了鉴定fox 1启动子调控元件，在转基因衣原体菌株（2A 38）中产生了插入文库，所述转基因衣原体菌株（2A 38）含有由fox 1启动子驱动的芳基硫酸酯酶（ARS）报告基因。选择对低铁条件有缺陷反应的突变体进行进一步研究。在这些菌株中，鉴定了含有被破坏的femu 2基因的菌株。femu 2基因产物对fox 1启动子的激活通过使用RNA干扰沉默femu 2基因来证实。在铁缺乏条件下，3个femu 2 RNAi转基因株系（IR 3、IR 6和IR 7）的ARS报告基因活性分别下降了84.3%、86.4%和88.8%。此外，两个femu 2突变体和RNAi转基因株系的RT-PCR分析表明，显着降低内源fox 1基因的转录丰度在铁缺乏条件下。氨基酸序列分析的femu 2基因产物确定了三个潜在的C2 H2锌指（ZF）基序和核定位研究表明，FEMU 2是本地化的细胞核。此外，使用PCR介导的随机结合位点选择鉴定了潜在的FEMU 2结合位点（（G/T）TTGG（G/T）（G/T）T）。两者合计，这一证据表明，FEMU 2参与上调铁缺乏细胞中的fox 1基因。
Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.
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