Study On The Role Of S-Layer Protein On The Cell Wall In The Formation Of Microcystis

Microcystis water-bloom has been recognized as one of the environmental hazards worldwide.there is no effective method to control it.Researches demonstrated that colony-formation be an important factor for Microcystis to get competitive advantage over other algae in the water.So making sure the molecular mechanism of colony formating has important significance to grasp the rules of Microcystis water-bloom outbreak.Our former researches found that the surface(S-layer)on the cell wall of unicellular Microcystis strain was very different from that of colony strain.Results of TEM and SDS-PAGE revelled that colonial Microcystis had a plenty of S-layer.However,the function of S-layer on colony formation needs more evidence to confirm.In this research,we cloned the S-layer gene of Microcystis into unicellular cyanobacteria Synechocystis PCC6803 to verify the function of the gene in colony formation.The main content in this paper is as follow:1.Construction of transgenic plasmid:We amplified the S-layer gene from Microcystis aeruginosa PCC7806 by PCR.Then we combined the S-layer gene,marker gene and DNA fragment of Synechocystis PCC 6803 into a plasmid vector pBS-Ppet with promoter petE which can be induced by Cu2+,to construct a recombinant plasmid pBS-S-p?.At the same time,we constructed a vector pBS-p? without S-layer gene as the control.2.Screening,identification,gene expression and phenotype analysis of mutant.?We transformed the plasmids above into Synechocystis PCC6803.We used antibiotic to screen and PCR to identify mutants.?The target gene expression was induced by Cu2+.The mutants with target gene formed colonies in several days,while there is no colony formation in the control strains.?The gene expression detected by fluorescence quantitative RT-PCR showed that much higher RNA level of S-layer appeared in the mutant than the control.?the analysis of total protein by SDS-PAGE showed a new protein band(S-layer protein)in the lane of mutant strain induced by Cu2+,which was not exist in the control or wild type.3.Observation of cell wall structure:The surface structure of mutant strain and w ild type were observed by TEM.The photos of TEM showed a thick layer of protein on the surface of the mutant.This result confired that expression of the S-layer improved the colony formation.Combining the previous results,we could come to the conclusion that S-layer could promote colony formation of Microcystis.