Gandhimathi Krishnan, Karthi Sellamuthu, Manimaran Paramasivam, Varalakshmi Perumal, Ashokkumar Balasubramaniem
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India.
J Biosci. 2015 Jun;40(2):257-68. doi: 10.1007/s12038-015-9512-x.
Riboflavin transporters (rft-1 and rft-2), orthologous to human riboflavin transporter-3 (hRVFT-3), are identified and characterized in Caenorhabditis elegans. However, studies pertaining to functional contribution of rft-2 in maintaining body homeostatic riboflavin levels and its regulation are very limited. In this study, the expression pattern of rft-2 at different life stages of C. elegans was studied through real-time PCR, and found to be consistent from larval to adult stages that demonstrate its involvement in maintaining the body homeostatic riboflavin levels at whole animal level all through its life. A possible regulation of rft-2 expression at mRNA levels at whole animal was studied after adaptation to low and high concentrations of riboflavin. Abundance of rft-2 transcript was upregulated in riboflavin-deficient conditions (10 nM), while it was downregulated with riboflavin-supplemented conditions (2 mM) as compared with control (10 meu M). Further, the 5'-regulatory region of the rft-2 gene was cloned, and transgenic nematodes expressing transcriptional rft-2 promoter::GFP fusion constructs were generated. The expression of rft-2 was found to be adaptively regulated in vivo when transgenic worms were maintained under different extracellular riboflavin levels, which was also mediated partly via changes in the rft-2 levels that directs towards the possible involvement of transcriptional regulatory events.
与人类核黄素转运蛋白-3(hRVFT-3)直系同源的核黄素转运蛋白(rft-1和rft-2)已在秀丽隐杆线虫中得到鉴定和表征。然而,关于rft-2在维持体内核黄素水平稳态及其调控方面的功能贡献的研究非常有限。在本研究中,通过实时PCR研究了rft-2在秀丽隐杆线虫不同生命阶段的表达模式,发现从幼虫到成虫阶段是一致的,这表明它在整个生命周期中都参与维持动物体内核黄素水平的稳态。在适应低浓度和高浓度核黄素后,研究了rft-2在整个动物mRNA水平上可能的表达调控。与对照(10μM)相比,在核黄素缺乏条件(10 nM)下,rft-2转录本的丰度上调,而在核黄素补充条件(2 mM)下则下调。此外,克隆了rft-2基因的5'调控区,并构建了表达转录rft-2启动子::GFP融合构建体的转基因线虫。当转基因线虫在不同的细胞外核黄素水平下维持时,发现rft-2的表达在体内受到适应性调控,这也部分地通过rft-2水平的变化介导,这表明可能涉及转录调控事件。