A Preliminary Study On Extracellular Vesicles Of Chlamydomonas Reinhardtii

Extracellular Vesicles(EVs)are nanoparticles with membrane structure secreted by cells.They are rich in substances,including nucleic acids,proteins,liposomes,etc.,and can shuttle between cells to achieve substances.Delivery,information exchange.Current research mainly focuses on higher animal and plant cells.Unlike higher biological cells,which have a stable internal environment,lower single-cell microalgae live in extremely complicated external environments.Little is known about its extracellular vesicles,and it is likely that It is a neglected but very important microalgae information exchange method,perhaps the most primitive way of communication for living things.This research aims at the current situation of microalgae extracellular vesicles with little research space and large research space,which has potential research value and practical significance.A suitable method for harvesting extracellular vesicles from the supernatant of microalgal cells was explored,and the harvested extracellular vesicles were characterized.Taking Chlamydomonas reinhartii as the main research object,this paper explored the extracellular vesicles of Chlamydomonas reinhartii,which were harvested under nitrogen stress and different growth stages,and their main components,including miRNA and protein.These findings indicate that the solubles in EV may be the result of special sorting by the sorting mechanism,and EV may be an important part of the information exchange between microalgal cells and play an important role in adapting to the changing aquatic environment.The main results of this paper are as follows:(1)The ultracentrifugation method and the PEG polymer precipitation method were compared to harvest microalgae extracellular vesicles.It was determined that ultracentrifugation was more suitable for the collection of microalgae extracellular vesicles.The results of extracellular vesicles harvested from a variety of microalgae indicate that both prokaryotic and eukaryotic microalgae can secrete extracellular vesicles,and microalgae seem to be able to secrete extracellular vesicles generally.The particle size results show that the average particle diameters of cyanobacteria PCC6803-EV and Chlamydomonas reinhardtii CC124-EV are 94.68 nm and 120.1 nm,which are consistent with the size of exosomes(30-200 nm),but they are more likely to be considered as a type of cells with small particle sizes External vesicles,called small extracellular vesicles(sEV).Chlamydomonas reinhartii EV tracer results show that Chlamydomonas reinhartii EV can enter recipient cells,which provides a basis and possibility for information exchange.(2)Small RNA sequencing results show that Chlamydomonas reinhardtii EV is rich in nc RNA,including(miRNA,rRNA,tRNA,snRNA,etc.).In-depth investigation of mi RNA components revealed the content and composition of mi RNA in extracellular vesicles and in cells.Not exactly the same,there is likely to be a sorting mechanism,and miRNAs are specifically sorted into EVs.In addition,it was found that the composition and content changes of Chlamydomonas reinhardtii EV-miRNA are closely related to cell growth and environmental stress.Using Chlamydomonas reinhardtii EV as a control,there was a significant difference in the composition of EV-miRNA in logarithmic phase.There were 11 significantly different miRNAs.It was found that many differentially expressed genes were related to cell growth.In addition,the composition of EV-miRNAs in Chlamydomonas reinhardtii also showed significant differences.There were 17 significantly different miRNAs.Many different genes were closely related to the cell response to stress environment.(3)Mass spectrometry analysis showed that Chlamydomonas reinhardtii EV was rich in proteins,and 2078 known proteins were detected,of which 1788 were quantifiable.It was further found that changes in EV protein composition and content are closely related to cell growth and environmental stress.The number of significant differentially expressed proteins is astonishing,involving important aspects such as material transport,metabolic processes,and photosynthesis.In addition,ESCRT-related proteins,which are essential for the transport of endosomal sorting complexes,have been discovered,which is essential for the specific sorting of extracellular vesicles.