Study On Screening,Identification And Action Mechanism Of M31 Protein,an Assembly Factor Of NDH-1 Complex,in The Cyanobacterium Synechocystis Sp.PCC 6803

In recent years,there have been many important advances in the biogenesis of chloroplast NDH-1 complex in higher plants.However,only Slr1097,a maturation factor of Ndh I subunit,and Ssl3829,an assembly factor for stabilization of Slr1097 and NDH-1 assembly intermediates are identified in cyanobacteria.Therefore,the study of assembly factors involving biogenesis of NDH-1 complex in cyanobacteria is important to understand the structure,functions and evolutionary change of NDH-1complex.In this study,NDH-CET impaired mutants were isolated by high-light screening method,and the cause of damaged NDH-CET activity was analyzed by reverse genetics with physiological and biochemical methods.We revealed the possible action mechanism underlying the effect of an assembly factor on NDH-1 complex,which greatly deepened the understanding of the biogenesis of NDH-1 complex.These related results were summarized as follows:(1)Two high light-sensitive mutants were isolated from Synechocystis 6803 transformed with a transposon-bearing library by using a high-light screening strategy.Both mutants impaired NDH-CET activity by analyzing the chlorophyll fluorescence and had a same tag in m31 gene by using PCR method.To confirm this possibility,we constructed a ?m31 deletion mutant and analyzed its NDH-CET activity by measuring chlorophyll fluorescence and P700 oxidation reduction kinetics.The results indicated that the absence of m31 gene impairs NDH-CET activity.(2)To reveal how the deletion of M31 impaired NDH-CET activity,we analyzed the accumulated level and assembly efficiency of NDH-1 complexes in thylakoids.The results indicated that the absence of M31 protein impairs the accumulation of NDH-1 complexes in the thylakoid membrane.We therefore concluded that the deletion of M31 protein leads to the destabilization of NDH-1 complexes in the thylakoid membrane and then results in impairing NDH-CET activity.(3)To reveal how M31 protein affected assembly of the NDH-1 complex,we investigated subcellular localization of M31 protein.The results suggested that M31 is a cytoplasmic protein.Further proteomics results suggested that the accumulation of NDH-1 complexes in the cytoplasm is also reduced and the absence of M31 protein resulted in a significant reduction of Ndh I.We therefore conclude that M31 influences the accumulation of NDH-1 complexes in the cytoplasm.(4)We further found that the M31 protein interacted with Ndh I subunit and its maturation factor,Slr1097.Therefore we speculated that M31 protein not only affects the maturation of Ndh I as an assembly factor,but also affects the accumulation of NDH-1 complex in the cytoplasm.In conclusion,this thesis identified an assembly factor of NDH-1 complex in cyanobacterium Synechocystis 6803 and revealed its possible action mechanism how M31 protein influence the assembly of NDH-1 complex.These findings will further provide novel understanding of the biogenesis of NDH-1 complexes in cyanobacteria.