Preliminary Study On The Mechanism Of Proliferating Cell Nuclear Antigen In Response To The Tempreature Tolerance In Ulva Prolifera O. F. Müller

Green tide,a marine disaster,has attracted people’s attention for more than ten years.As previous reported,green tide is mainly caused by the explosive growth of green alga,for example,Enteromorpha linza,Enteromorpha clathrata,Enteromorpha intestinalis and Ulva prolifer a(U.prolifera).Among these species,U.prolifera has been proven to be the main species of green tide,and the reasons for its continouslly outbreak could be summarized as following,such as the eutrophication of water,the suitable temperature of Yellow Sea and so on.In addition,U.prolifera is widely tolerant to the abiotic stress,including temperature,salinity and light,while the molecular mechanisms of which are diverse.However,due to the lack of commercially antibodies and the incomplete genome database,the limitation of technology caused by the characteristics of U.prolifera,the underlying mechanisms of U.prolifera responsing to the abiotic stress is still unclear.In this study,we set different cultural temperature to simulate the influence of the temperature changes on the growth of U.prolifera.The results suggested that the relative growth rate was increased following the enchanced temperature,and the contents of chlorophyll a and chlorophyll b were both significantly increased in U.prolifera cultured at 36℃ for four days.At the same time,the transcriptome sequencing results indicated that high temperature induced the expression of genes related to some metabolism pathways including photosynthesis and carotenoid synthesis pathway,but also inhibited the expression of genes related to polysaccharide degradation,glutathione metabolism and protein metabolism pathways.These data suggested that accumulating the polysaccharides and shutting down the metabolism of proteins might be the strategy for U.prolifera to adapt to the high temperature tolerance.On the other hand,low temperature had no significant effect on the expression of genes mentioned above,which was consistent with the results that the relative growth rate kept stable in U.prolifera under low temperature.Furthermore,to investigate the underlying molecular mechanism of U.prolifera to resist to the temperature stresses,we successfully cloned cell proliferation-associated genes based on the transcriptome analysis.Proliferation cell nuclear antigen(PCNA),mitotic-specific cyclin A(CyclinA)and mitogen-activated protein kinase(MAPK)were amplified through polymerase chain reaction,and the amino acid sequences of these genes were compared with that from other alga or plants.The results of comparison analysis indicated that the similarities of these genes were small between U.prolifera and other species.Additionally,CyclinA was proved to be the target binding protein of PCNA through yeast two-hybrid and immunoprecipitation assays.Subsequently,quantative real-time polymerase chain reaction(qRT-PCR)and Western blot analysis were used to investigate the role of PCNA-CyclinA-MAPK axis in U.prolifera under temperature stresses.The results demonstrated that PCNA and CyclinA levels maintained at a high level under low temperature.However,the expression of PCNA and CyclinA fluctuated under low temperature stresses,which was that both genes were up-regulated firstly and then down-regulated.Interestingly,the levels of phosphorylated ERK1/2,which was defined as MAPK3/6 in plants,were consistent with the expression trend of PCNA under different temperature stresses.Since the protoplasts isolated from the tubular alga could be regenerated and further develop into a new strain,we used enzymatic hydrolysis buffer to obtain vigorous protoplasts.Furthermore,Evans blue staining and fluorescent whiten staining analysis were conducted to explore the influnce of temperature on the germination of U.prolifera.The data indicated that high temperature enhanced the rates of dead protoplasts while inhibited the division of protoplasts within cell wall.However,low temperature had no significant effect on the survival and division of protoplasts.Additionally,the activity protoplasts were reduced and the cell contents were precipitated after incubated with PCNA inhibitor.Moreover,PCNA inhibitor inhibited the regeneration of protoplasts in a dose-dependent manner.We also explored the upstream genes of PCNA based on the bioinformatics analysis,and the results showed that PCNA gene contained the binding sites of miR-2916 at-552 to-772.To verify the combination between miR-2916 and PCNA,qRT-PCR assays were conducted and the results of which demonstrated that the levels of miR-2916 were negatively correlated with the expression of PCNA.So the data above suggested that down-regulated miR-2916 participated in the process of U.prolifera resisting to temperature stresses partly through regulating PCNA expression.In conclusion,the above results supported the common view that U.prolifera was derived from the microscopic propagules.We suggested that low-expressed miR-2916 in the microscopic propagules from the sea bottom mud regulated the expression of PCNA,thereby activating the CyclinA-MAPK signaling pathway,which contributed to the proliferation and division of U.prolifera.Moreover,when the temperature of the surface seawater bagan to rise,the vegetative cells and germ cells from the whitish segment accelerated their proliferation and division process through up-regulating PCNA levels.In general,it’s the first time to explore the possible mechanism of overwintering and over-summer action in U.prolifera,at the same time,the study provided a new insight for the prevention and treatment of green tide by target PCNA.