| Cyanobacteria is the oldest and only prokaryote known for photosynthesis and oxygen release.It fixes about 10% of carbon dioxide in the atmosphere yearly through photosynthesis,which has important research value in the global carbon cycle,biological clock and biological evolution.Cyanobacteria have a variety of cell morphology,and their growth and morphology are closely related to physiological functions.The structure and composition of the cell wall of cyanobacteria are particular.They are classified as gram-negative bacteria because of their outer membrane structure,but the structural characteristics of their peptidoglycan layer are more similar to those of gram-positive bacteria.In addition,cyanobacteria also have their own unique cell wall synthesis regulation mechanism.Due to the complexity of cell structure,the research on how cyanobacteria maintain cell morphology and precisely control the formation of the cell wall during cell growth and division is still immature.Synechococcus elongatus PCC 7942 is a rod-shaped single-cell cyanobacteria,and also a model material for studying the physiological function and morphogenesis of cyanobacteria.In this research,S.elongatus PCC 7942 was taken as the research object,and the genes related to cell morphogenesis were discovered by constructing a high-throughput protein marker system,and an annotated gene Synpcc7942＿RS00055,which was conserved in cyanobacteria,was identified,which proved that the gene coding protein had an important regulatory function in the growth and division process of S.elongatus PCC 7942,belonging to a new type of cell division regulatory protein.The main research contents of this thesis are as follows:(1)Construction of high-throughput protein subcellular localization technology of S.elongatus PCC 7942: A high-throughput cloning system suitable for S.elongatus PCC 7942 was constructed based on Golden Gate method,and a high-throughput "traceless" protein fluorescence marker of S.elongatus PCC 7942 was successfully established by introducing cod A gene,and a microscopic imaging method suitable for S.elongatus PCC 7942 was established.First,96 genes were tested,and a high-throughput operation was carried out to realize the subcellular localization imaging of fluorescence labeling of 56 proteins,and the proteins located in different cell structures were assigned.On this basis,an unknown functional protein Synpcc7942＿RS00055 located near the Z ring of cell division center was screened,which laid a good foundation for further study of the function of this protein,Hereafter in this study,we name this protein as Ccd R(Cyanobacterial Cell Division Regulator).(2)Analysis of subcellular localization of Ccd R protein in Polycystis elongata: By introducing green fluorescent protein m Neon Green at the C-terminal,fluorescence labeling and microscopic imaging of the non-annotated functional protein Ccd R were carried out.Through super-resolution microscopy and Confocal laser scanning microscope,it was observed that Ccd R-m NG exists as dots near the Z-ring of the cell division center and at both polar of the cell.Combined with dynamic analysis,it was found that when cells do not undergo division,proteins are dispersed on the cell membrane;during cell division,proteins quickly move and aggregate to the center of cell division.In addition,the expression level of proteins is related to the cell growth cycle.When the cell is in the early phase of logarithmic growth,which is the most vigorous period of division,the protein expression level is high and located near the Z ring of the cell division center;When the cell is in the late or stable phase of logarithmic growth,the protein expression level is low and mostly at the cell polar.Based on the subcellular localization characteristics of Ccd R protein,it is speculated that Ccd R may be related to cell growth and division.(3)Study on the Physiological Function of Ccd R Protein in S.elongatus PCC 7942: ccd R gene knock out Mutant Strain of S.elongatus PCC 7942 was constructed using Physiological genetics Methods(Δccd R),and further introduce the ccd R gene into the NSI neutral site of the mutant strain to obtain a complementary mutan(Δccd R::ppsb A-ccd R).The growth curve test shows that the growth rate of the Δccd R strain is lower than that of the wild-type strain,and the replacement strain can restore the growth level of the strain.Meanwhile,the cell morphology of the Δccd R strain undergoes significant changes,with a smaller cell diameter and the formation of fibrous cells,which may be due to cell division but not separation.Observing with a transmission electron microscope,the cell division membrane formation zone of the Δccd R strain is different from that of the wild-type,and asymmetric membranes were observed in the complementary strain.In summary,the knockout of the ccd R gene affects the growth,division,and cell morphology of S.elongatus PCC 7942.(4)Study on the Physiological Function of Ccd R Protein in S.elongatus PCC 7942: FLAG tag was labeled at the C-terminal of Ccd R protein.After incubation with the anti-FLAG antibody,28 proteins specifically bound to Ccd R were identified by affinity chromatography-mass spectrometry,including peptidoglycan synthetase Fts I and cell rod-shaped morphology-related protein Rod Z.Using bacterial two-hybrid,interaction analysis was conducted on 17 proteins,including specific binding proteins and cell division related proteins.The results were all negative,indicating that there is no direct interaction relationship between Ccd R protein and cell-division related proteins.(5)Analysis of the regulatory mechanism of ccd R gene in S.elongatus PCC 7942: using transcriptome technology,the gene expression level of Δccd R mutant and wild-type strain was analyzed and compared,and 184 differentially expressed genes were identified,of which 76 genes were significantly up-regulated in the deletion mutant,and 108 genes were significantly down-regulated.These genes are mainly related to thylakoid membrane structure and photosynthesis,and include the gene bcs A related to bacterial cellulose.Further using Ch IP-seq sequencing technology,233 gene fragments that may interact with Ccd R were obtained.Through joint analysis with transcriptome data,19 related genes were identified,which are mainly related to protein synthesis,gene transcription and translation.Through Peak reads analysis,the most likely Motif sequence to bind to the Ccd R protein were identified.The Motif sequence predicted in Ch IP-seq was identified as a specific binding sequence of Ccd R protein through MST technology.Based on the above analysis,it is speculated that Ccd R is a global regulatory protein,which is related to bacterial growth,division and peptidoglycan layer synthesis.(6)Recombinant expression of gene ccd R in E.coli and Pichia pastoris and its regulation on cell morphology: In order to clarify the universal rule of Ccd R protein regulating bacterial morphogenesis,based on expression vector p ET-22 b and p MY69,a recombinant plasmid containing gene ccd R was constructed,which was introduced into E.coli BL21 and Pichia pastoris PPY12 for recombinant expression,and the recombinant expression strain was obtained.Dyes DAPI and FM4-64 were used to stain and image the chromatin and extracellular membrane structure of the recombinant bacteria.The statistical results showed that the morphology of the strain overexpressing ccd R changed,a large number of round cells appeared,and the cell diameter significantly increased.Moreover,the cell size of Pichia pastoris was significantly increased.Thus,it is further proved that Ccd R protein plays a universal role in the maintenance of cell morphology of bacteria and yeast.To sum up,on the basis of constructing a high-throughput protein subcellular localization system,this paper identified an uncommented protein,Ccd R,which is closely related to the cell growth and division of S.elongatus PCC 7942.Through molecular genetics,fluorescent labeling and multi-omics technology,the biological function of Ccd R in the growth and division of S.elongatus PCC 7942 was further clarified,.Through heterologous expression,the functional characteristics of this novel division-related protein,which is universally involved in the growth,division,and cell morphology regulation of rod-shaped bacteria,were verified. |
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