| 论文摘要： |
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 Cre-miR914 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 bioinfounatics 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 (cw15), while RPL18 overexpression lines had a higher growth than the wild-type line (cw15). Cells vitality (photosynthesis activity) experiment under stress also demonstrated that Cre-miR914 overexpression lines had a lower vitality than the wild-type line (cw15), 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 (cw15), 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. |
