链状亚历山大藻不同生长状态差异表达microRNA的筛选和验证  

The Screen and Verification of Differentially Expressed MicroRNAs Under Different Growth Conditions in Alexandrium catenella

作　　者：耿慧利 隋正红[1] 任源源[1] 杜青伟 刘源[1] 

机构地区：[1]中国海洋大学海洋生物遗传学与育种教育部重点实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2016年第1期64-71,共8页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(41176098);山东省自然科学基金重点项目(ZR2011DZ002)资助~~

摘　　要：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。As a class of relatively conservative regulator,accumulated reports demonstrated that miRNAs play an important role in a large variety of biological and metabolic processes,including cell proliferation,organ maturation,signal transduction and response to stress.Dinoflagellates is a kind of important organisms that can cause harmful algal blooms（HABs）.Alexandrium catenellais a representative of dinoflagellates that is widely spread all over the world and has the ability of producing PSTs.In this paper,two differentially expressed microRNAs（osa-miR2876 and rgl-miR5139）were selected from constructed small RNA libraries of A.catenella under the lag and the logarithmic phases.Five conditions including the lag phase and the logarithmic phase under f/2medium,high N,high P and high Mn induced logarithmic phase were designed for expression detection via qPCR technique.The study established a poly（A）-tailing qPCR method with SYBR Green I to detect the differential expression of microRNA under different growth conditions in A.catenella.5.8srRNA gene was used to normalize the expression of microRNAs.The expression level of microRNA under the logarithmic and induced logarithmic（eg.high N,high P and high Mn induced logarithmic phase）phases were all lower than those under the lag phase.The lowest expression level was shown in the loagrithmic phase under high Mn.The expression level in the lag phase was 18.63（for osa-miR2876）and 14.12（for rgl-miR5139）,respectively of that under high Mn condition.microRNA regulated gene expression via a negative pattern.The result of real-time PCR was consist with highthroughput sequencing.osa-miR2876 and rgl-miR5139 in the logarithmic phase were both down-regulated,which suggested that the target genes of two microRNAs involved,eg.DNA replication,repairing,the metabolism of purine and pyrimidine,the metabolism of RNA polymerase,may be activated.The activation of the above physiological processes were all benefit for the proliferation of the cells.Our results suggested tha

关 键 词：链状亚历山大藻 MICRORNA qPCR 差异表达 

分 类 号：S917.3[农业科学—水产科学]