A Preliminary Study On Chloroplast Development Of Euglena Under Photoresponse

Chloroplast-related research has been a focused topic for many years,such as the structure and function of chloroplasts,the origin and development of chloroplasts,and improvement of photosynthetic efficiency.The structure and functions of chloroplasts have been thoroughly investigated.However,the research on the origin and development mechanism of chloroplasts is ambiguous.The origin of chloroplasts is widely recognized as the symbiotic hypothesis proposed by Mereschkowsky in 1905.For the development of chloroplasts,there have been no well-developed mechanisms so far,probably due to its complexity.In addition,for Euglena gracilis,a very old green protist with special chloroplast membrane structure,has the unique property of stably surviving in the absence of functional chloroplasts.And there are natural colorless algae,such as Astasia longa,which contains an indispensable residual plastid structure and around half plastid genome.So Euglena can be regarded as the best material to research development of chloroplasts and its molecular communication with nucleusTherefore,in this study we performed the dark synchronized culture of the colorless wild-type A.longa,E.gracilis,and the bleached mutant strain B2 derived from E.gracilis,and then obtained and analyzed the information of transcriptome and metabolome under light-stimulation by the Hiseq Xten sequencing platform and the GC-MS method,respectively.The aim is to discover the genetic information and metabolic pathways related to development of chloroplasts during the light response of Euglena.And in order to establish a stable and efficient method of genetic transformation in E.gracilis,the methods such as glass bead-assisted transformation,particle gun-mediated(biolistic),and electroporation were atempted.The RNA-seq raw data were filtered and assembled by Trinity to show that A.longa,E.gracilis & B2 had 148,385 and 151,809 transcripts,respectively.Using DEseq for differential expression analysis,A4(A.longa after light transition for 4 h)were identified that had 13 obvious down-regulated genes compared with A0(A.longa before the light treatment),and 28 obvious up-regulated genes.And 75 clear down-regulated genes were observed in A8 compared to that of A0,with 343 up-regulated genes.After 12 h of light,more differentiallyexpressed genes were found,with significantly down-regulated 258 genes,and up-reguated752 genes.In contrast,E.gracilis had only 30 and 32 genes which significantly down-regulated and up-regulated after 4 h of light-stimulation compared to that of E0.For the bleached mutant,B2 train had only 27 and 35 genes significantly down-regulated and up-regulated after 4 h of light treatment compared to that of B0.The GO annotation and KEGG functional enrichment results of A.longa,E.gracilis and B2 showed that there were no clear differences between 4 h and 8 h of light-stimulation,while A.longa was enriched in photosynthetic pathway and chloroplast-related differential genes at 12 h light-stimulation.The results of Metabolomic of E.gracilis & B2 showed that light-stimulation significantly affected the intracellular metabolic compositions and contents.The green E.gracilis increased the content of some metabolic components such as Phosphoric acid after light-stimulation,but bleached mutant B2 reduced the content of some metabolic components after light-stimulation.And there was a significant difference between the speed of E.gracilis and B2 in response to light-stimulation.An antibiotic-sensitive experimental study of E.gracilis found that the cells were resistant to most antibiotics but not to G418 and Zeocin.The results of genetic transformation were only successfully transformed by particle gun-mediated(biolistic)method,but with poor efficiency and stability.In summary,this study is mainly to conduct preliminary research and analysis of the response mechanism of Euglena under light-stimulation,and to establish a stable and efficient genetic transformation method for further research and gene function verification.We hope to more clearly understand the relevant mechanisms in the development of chloroplasts and the bidirectional regulation communications between chloroplasts and nucleus,thus providing a theoretical basis and reference value for synthesis of chloroplasts in vitro and in vivo.