Exploring The Dynamic Changes Of Proteome And The Effects Of Epiphytic Bacteria During Nostoc Flagelliforme Response To Rehydration

Terrestrial hairy algae,commonly known as Nostoc flagelliforme,is an autotrophic nitrogen-fixing cyanobacterium.It grows mainly in arid and semi-arid regions where annual rainfall is scarce.In this environment of alternating wet and dry conditions,Nostoc flagelliforme(N.flagelliforme)has evolved a strong ability to resist stress,but the molecular mechanism of drought tolerance and rehydration remains unclear.Proteomics research has found some differentially expressed proteins during dehydration and rehydration of N.flagelliforme.However,these studies have focused on changes in intracellular proteins and ignored the analysis of extracellular proteins.Extracellular proteins play a critical role in maintaining cell structural stability and physiological activity.They will inevitably affect the rehydration of N.flagelliforme.In addition,the epiphytic bacteria closely linked to the N.flagelliforme in the environment.Presumably it has a symbiotic relationship with N.flagelliforme,but its effect on the rehydration of the N.flagelliforme has not been deeply studied.Therefore,in order to further reveal the molecular mechanism of rehydration of N.flagelliforme,this thesis will explore the changes of intracellular and extracellular proteins during the rehydration process of N.flagelliforme based on non-standard quantitative proteomics technology.Meanwhile,this work will also compare the different proteome of N.flagelliforme under the condition of epiphytic bacteria or not.In this study,the intracellular and extracellular proteins during the rehydration of the sterilized and unsterilized groups were collected separately.8 rehydration time points were selected for each group,which were 0 h,0.25 h,0.5 h,1 h,3 h,8 h,15 h,24 h.Based on the label-free quantitative proteomics technology,dynamic change of protein during the rehydration of N.flagelliforme was explored.In the unsterilized group of rehydration at 8 time points,all the identified proteins were subjected to trend cluster analysis,and divided into 4 categories.GO annotation analysis shows that their functions mainly include defense response,photosynthesis,protein synthesis and degradation,metabolism,signal regulation and transport system.The results showed that some proteins related to rehydration of N.flagelliforme only played a role in the cells.For instance,the ATP-dependent zinc metalloproteinase FtsH showes an upward trend inside the cells,while it was not detected outside the cells.Some proteins differ only extracellularly,such as the type ? pilus assembly protein PilM.Some other proteins are expressed differently inside and outside the cell.proteins containing the bacterial Ig-like domain and the vWFA domain have a downward trend in the cells and high expression outside the cells.They is highly homologous to perforin and may be related to the destruction of the sugar chain of the polysaccharide layer,which is beneficial to the water absorption of N.flagelliforme.And water stress proteins have a downward trend inside the cells and high expression outside the cells.,closely related to the rehydration of N.flagelliforme.Besides,it has been found that the proteins related to drought tolerance of N.lagelliforme showed a downward trend in the cells,while gradually increased outside the cells,such as ribosome hibernation promoting factor(HPF)and cation transport regulator ChaB.They may be secreted outside the cell due to the release of drought.In the comparative proteome study of the sterilized group relative to the unsterilized group,229 significant differentially expressed intracellular proteins were identified(P<0.05),of which 31 were extremely significant(P<0.001).Their functions mainly involve defense systems,protein synthesis and degradation,and photosynthesis.89 significant differential proteins were identified in extracellular proteins(P<0.05),of which 4 were extremely significant(P<0.001),Their functions mainly involved defense system and signal regulation.Among them,the alkyl hydroperoxide reductase AhpD has a high extracellular content at the beginning of rehydration.It may be secreted into the extracellular space during drying to protect the body from damage by scavenging free radicals.In addition,proteins are up-regulated during rehydration in the unsterilized group,such as the RTX toxin related,phycocyaninase and glycosidase.They are secreted to the outside.Among them,phycocyanase may provide carbon and nitrogen sources for epiphytic bacteria and N.flagelliforme due to the degradation of phycocyanin,and the RTX toxin related secreted by N.flagelliforme.It may be related to the increasing number of epiphytes in the rehydration process.Through this study,we further explored the mechanism of rehydration of N.flagelliforme.During rehydration of N.flagelliforme,the redox protein,HPF and alkyl hydroperoxide reductase AhpD and drought-tolerant proteins were relieved of drought,and secreted outside the cell.Among them,the alkyl hydroperoxide reductase AhpD plays an antioxidant role in the outer membrane of the body to protect the body during drought.The ATP-dependent zinc metalloproteinase FtsH is abundantly expressed to degrade and repaire damaged proteins in vivo.Beside,some regulation Osmotic proteins such as ChaB,OmpR and OsmY are secreted outside the cell to maintain osmotic balance.The protein associated with RTX toxin is secreted outside the cell and may be related to the increasing number of epiphytes in the rehydration process.The relationship between the two needs further study.