Functional Characterization Of The S2P Proteases In Synechocystis Sp.PCC 6803 Under Stress Response

The development of new energy sources is becoming more and more important as the energy demand increases rapidly.Application of microalgae in biomass production is one of the promising strategies.Howerve,the stress caused by accumulation of biomass will affect the growth of microalgae as well.Synechocystis sp.PCC6803 is a model organism to study abiotic stress response,and it has been applied in the development of biomass.The intracellular proteolysis regulated by S2P protease is considered to be an important signal transduction mechanism involved in activating gene expression during acclimation to stress.Thus,functional characterization of S2P protease in Synechocystis sp.PCC6803 will not only provide a theoretical basis for the study of stress acclimation,but also lay foundation for the development of engineering microalgae which could tolerate abiotic sress.There are four S2P proteases in Synechocystis sp.PCC6803.This thesis studied two of them,Sll0528 and Slr0643.The Sll0528 overexpression mutant OE0528 was constructed,and along with the deletion mutant?sll0528,they were used to explore the function of sll0528 in response to multiple stresses.Then the transcriptional sequence data of slr0643 knockout mutant?slr0643 under glucose mixotrophic condition was analyzed to investigate the mechanism of slr0643 in response to glucose mixotrophism.(1)Through construction of recombinant plasmid and homologous transformation of Synechocystis,the overexpression element km~r+psbA2 promoter+sll0528 was inserted into the neutral site between slr2030 and slr0230 in Synechocystis sp.PCC6803 to create the sll0528 overespression mutants.Totally,six overexpression mutants OE0528 were obtained.The expression level of sll0528 was significantly higher than the wild type,indicating by RT-qPCR.Growth of OE0528 mutant was similar with wild type under normal physiological conditions,suggesting that overexpression of sll0528 had little effect on the normal growth of Syncchocystis sp.PCC6803.(2)The physiological properties of the sll0528 knockout mutant?sll0528 and sll0528overexpression mutant OE0528 was investigated under ammonium salt stress.Growth of?sll0528 was hampered under 120 mM ammonium chloride,and the chlorophyll and phycobiliprotein content were lower than wild type.While the growth trend of OE0528 is obviously better than wild type under 180 mM ammonium chloride,with higher chlorophyll and phycobiliprotein content than wild type.Lack of sll0528 leading to photosynthetic system damage under ammonium stress may be one of the reasons that?sll0528 can not grow under120 mM ammonium chloride stress.While in the OE0528 mutant,overexpression of sll0528may protect or repair the photosynthetic system damage under higher concentration of ammonium chloride,render OE0528 to grow better than wild type in 180 mM ammonium chloride.(3)Under osmotic stress as 0.5 M sorbitol stress in medium intensity light condition,the growth rate of OE0528 was higher than wild type,and the content of chlorophyll and phycobilis protein were also higher than wild type.It is speculated that sll0528 may regulating the expression of pigment related genes in photosynthetic system when OE0528response to osmotic stress.The growth trends of OE0528 mutant under NaCl and high light stress were similar with wild type,suggesting overexpression of sll0528 did not result in higher resistance of mutant than wild type upon these stresses.(4)Under 2.5 mM glucose mixotrophic condition,the growth rate and glucose utilization rate of slr0643 knockout mutant?slr0643 were lower than wild type,indicating the importance of functional Slr0643 for Synechocystis to adapt to glucose mixotrophism.The transcriptome sequencing data of mutant?slr0643 and wild type under photoautotrophism and glucose mixotrophism was analysed and compared.It was found that wild type adapted to glucose mixotrophism through reducing photosynthesis,increasing nitrogen metabolism and raising iron ion intake.While mutant?slr0643 could not adapte to glucose mixotrophic,the osmotic pressure correlative genes are up-regulated,the iron ion intake was decreased,and decrease of photosynthesis was more remarkable.Thus,knockout of slr0643 resulted in decrease of glucose utilization,depression of iron uptake and photosynthesis in mutant?slr0643.Moreover,compared with wild-type,higher osmotic pressure related genes was observed indicating higher osmotic pressure existed in mutant.The iron ion intake and carbon metabolism pathway were also down-regulated in the mutant,which may be part of the reasons why the growth rate of mutant is lower than that of wild type.