从链状亚历山大藻(Alexandrium catenella)延迟期、对数期2个小RNA文库中选择了2个差异表达的microRNA(osa-miR2876和rgl-miR5139),设计f/2培养下延迟期、对数期和高氮、高磷、高锰对数期5个条件,用qPCR的方法对其表达进行了检测。研究建立了以5.8srRNA为内参基因,以SYBR Green I为荧光染料,用加尾法荧光定量PCR检测microRNA在不同生长状态相对表达的方法。microRNA的表达量在对数期和诱导下对数期均低于延迟期,高锰条件下对数期均为最低,且延迟期的表达量分别是高锰条件表达量的18.63和14.12倍。microRNA负调控靶基因的表达,而osamiR2876和rgl-miR5139在对数期均为下调表达,说明其靶基因参与的细胞生理过程:DNA复制、修复、嘌呤、嘧啶碱基的代谢、RNA聚合酶的代谢等过程呈现激活状态,而上述生理过程的激活均有利于藻细胞增殖。这些结果表明:osamiR2876和rgl-miR5139可能在链状亚历山大藻的快速分裂和增殖中发挥着非常重要的调控作用。本文研究结果为甲藻兼性营养型正确性提供了进一步的证据,同时rgl-miR5139在细胞快速生长阶段的下调表达提示在该阶段下异养营养型可能占优势。本研究说明了microRNA在链状亚历山大藻快速生长过程中可能发挥着重要的调节作用。也为其他物种microRNA的研究和检测提供了快速简便、特异性好的研究方法。
Based on the transcriptomic sequencing, three genes of Alexandrium pacificum encoding ubiquitin(UBI), telomerase(TEL) and glycine-rich protein(GRP) relating to cell division were isolated and characterized. The full-length cD NA of GRP was obtained through approach of rapid amplification of c DNA ends(RACE). Four conserved domains, including DNA-and RNAbinding sites or motifs, cold shock domain at the N-terminal, and zinc-finger structure of CCCH type at the C-terminal were idenrtified. Phylogenetic analysis revealed that the deduced amino acid sequence of GRP tends to cluster together with proteins harboring cold shock domain. The expressions of these three genes were analyzed with quantitative PCR(qPCR). It was found that the expressions of these three genes at the logarithmic growth phase and induced logarithmic growth phase were all higher than those at lagging growth phase(P < 0.05). The expression patterns of these three genes were coincident with the proliferative capacity of the algae, e.g., displaying increased expression level at log and induced growth phases. The functions of these genes and their possible roles during harmful algal blooms(HAB) were discussed.
