摘要：
Heat shock is a common stress for life,while algae develops high efficient adaptation ability to heat shock during longtime evolution.Up to date,the researches about the mechanism of heat shock adaptation in algae focus just on physiological regulation and related coding genes,while there are few reports about non-coding genes on it.In the previous study,we found that Cre-miR914 were down-regulated significantly under multiple stresses (heat shock,UV-B and salinity) in Chlamydomonas reinhardtii through Q-PCR screening experiments,and bioinformatics analysis showed that the target gene of Cre-miR914 may be RPL18.But the functions of CremiR914 and its target gene in heat shock adaptation are unclear,this study addressed these issues through multiple experiments.In this study,we identified the target of Cre-miR914 through bioinformatics and degradome sequencing,and validated expression of Cre-miR914 and RPL18 under heat shock through Q-PCR.Then we constructed cell lines of Cre-miR914 overexpression and RPL18 overexpression for further study.And finally we performed stress adaptation experiments under heat shock stress to check the function of microRNA and its target in stress adaptation,which includes cell growth assay,cell vitality counting,reactive oxygen species (ROS) production and lipid peroxidation (MDA) measurements.Bioinformatics and degradome sequencing indicated the target of Cre-miR914 is RPL18;Q-PCR results showed that Cre-miR914 expression reduced under heat shock,but RPL18 expression increased,which confirmed our previous results of screening experiment.Then we got more than 3 cell lines with overexpressing of Cre-miR914 and RPL18.Further growth experiment under heat shock indicated that Cre-miR914 overexpression lines had a lower growth than the wild-type line (cwl5),while RPL18 overexpression lines had a higher growth than the wild-type line (cwl5).Cells vitality (photosynthesis activity) experiment under stress also demonstrated that Cre-miR914 overexpression lines had a lower vitality than the wild-type line (cwl5),while RPL18 overexpression lines had a higher vitality.The cell damage (ROS production and MDA content) experiments showed that there were more cell damages (ROS production and MDA content) in Cre-miR914 overexpression lines than the wild-type line (cwl 5),while that of RPL18 overexpression lines were lower than the wild-type line (cw15).These results illustrated that overexpression of Cre-miR914 reduced heat shock resistance ability in algae,while overexpression of RPL18 increased heat shock resistance ability.We maybe discovered a new regulation mechanism of heat shock adaptation in algae,in which Cre-miR914 and its target gene RPL18 are engaged in adaptation regulation to heat shock in Chlamydomonas reinhardtii.%高温胁迫是生物所面临的常见环境胁迫,因此在长期进化中生物逐渐进化出了对高温胁迫的高效适应能力.目前,有关藻类对高温胁迫的适应机制研究主要集中在生理调控及其相关的编码基因调控方面,而有关非编码基因对高温适应的调控尚无报道.在前期研究中,我们通过对衣藻细胞的定量PCR筛选和生物信息学分析发现,在多种胁迫处理后Cre-miR 914表达下调且其靶基因有可能是RPL18,但对它们的作用却不清楚.本研究中利用生物信息学结合降解组测序确定了Cre-miR 914的靶基因是RPL18,接着利用定量PCR验证Cre-miR 914及其靶基因的表达情况,发现Cre-miR 914表达在高温处理后明显下调,而RPL18表达明显上调,同时通过构建Cre-mi 914过表达株和靶基因RPL18过表达株,结合高温胁迫处理和抗性表型研究,发现Cre-miR 914过表达明显降低衣藻对抗高温胁迫能力,而靶基因RPL18过表达提高了衣藻对抗高温胁迫能力.本研究发现了一个microRNA参与调控藻类高温适应过程的分子机制,即衣藻通过调控Cre-miR 914及其靶基因RPL18表达参与了的高温胁迫适应过程.
