Screening And Functional Analysis Of Autotrophic And Heterotrophic Conversions Of Small RNA And Regulation Of Lipid Synthesis MicroRNAs In Auxenochlorella Protothecoides

With the rapid development of economy in modern society,traditional fossil fuels can no longer meet the world's increasing demand for energy.In order to cope with the energy crisis and the greenhouse effect brought by fossil energy,the development of clean and renewable bioenergy has drawn great attention from researcheres.Microalgae has been considered as a promising candidate for bioenergy production with its fast growth rate,high oil content,and no occupation of arable land.Auxenochlorella protothecoides is a single-cell eukaryotic green algae,which has high oil content and can be cultivated under both autotrophic and heterotrophic modes.Especially under heterotrophic conditions,A.protothecoides can accumulate a large amount of oil,therefore has great potential in the production of biodiesel.As the genetic transformation system of A.protothecoides is still under investigation,current researches on A.protothecoides oil accumulation were mainly focused on the biochemical regulation.To investigate whether miRNA is involved in the regulation of high yield oil accumulation under heterotrophic conditions in A.protothecoides,high-throughput miRNA sequencing was conducted in the present study,and the small RNA expression profiles of A.protothecoides under autotrophic and heterotrophic conditions were analyzed.The results were as followings:1.The subcellular structure of A.protothecoides was observed by transmission electron microscopy.Under autotrophic condition,the algal cells were filled with chloroplasts and contained only a few lipid droplets;while under heterotrophic condition,A.protothecoides cells were full of oil droplets and chloroplasts were not visible;which indicated that under heterotrophic condition,A.protothecoides accumulated large quantities of oil droplets.Further analysis showed that the lipid content of A.protothecoides reached 47.1%(470.9 g/mg DW)of its dry weight under heterotrophic condition,which was significantly higher than that(31.8%,318.5 g/mg DW)under autotrophic condition.2.High-throughput sequencing was used to analyze the expression profiles of small RNAs in A.protothecoides under autotrophic and heterotrophic conditions.8312780 and 4004888 Useful reads were obtained respectively.Their size distribution showed that the most frequent size length was 20 nt,and the first nucleotide preferred to be Uridine(U).Under autotrophic condition,173 known miRNAs and 61 new miRNAs were detected,While there were 166 known miRNAs and 40 new miRNAs identified under heterotrophic condition.Among them,126 known miRNAs and 25 new miRNAs were shared under both autotrophic and heterotrophic conditions.3.Differentially expressed miRNAs analysis shows that the expression of 18 miRNAs(all are Novel miRNA)were significantly varied between autotrophic and heterotrophic conditions.Compared to those under autotrophic condition,5 miRNAs were significantly up-regulated and 13 miRNAs were significantly down-regulated.Target prediction of these differentially expressed miRNAs revealed that the miRNAs in A.protothecoides have multiple target genes.GO enrichment showed that these target genes were mainly enriched in the metabolic process,membrane,organelles,catalytic activity and binding activity.KEEG pathway analysis showed that the target genes were mainly distributed in glucose metabolism,signal transduction,transportation and catabolism,amino acid metabolism.4.The 18 significantly differentially expressed miRNAs were verified by qRT-PCR.The results showed that 13 miRNAs could be detected,among which 7 miRNAs showed significant differences in expression level,with Nov-m0033-5p presented the largest difference.Compared with autotrophic condition,the expression level of Nov-m0033-5p in A.protothecoides was down-regulated by 71% under heterotrophic condition.5.Through the analysis of key enzymes in lipid synthesis pathway,it was found that compared with autotrophic condition,the DGAT gene under heterotrophic condition was significantly up-regulated,while CIS gene was significantly down-regulated,indicating that the lipid synthesis pathway was significantly enhanced during heterotrophic cultivation.6.Taken together the target gene analysis of Nov-m0046-3p,Nov-m0050-5p and Novm0071-3p,and the transcript level analysia of key enzymes in lipid synthesis,it can be speculated that under heterotrophic condition,the up-regulation of Nov-m0046-3p,Novm0050-5p and Nov-m0071-3p significantly reduced the expression level of their target genes,including enol phosphate pyruvate carboxylase,malic acid synthase and citrate synthase,and subsequently affected the direction of carbon flow in the algal cells,and promoted the lipid synthesis.In summary,this study revealed that miRNAs in A.protothecoides participate in the regulation of oil synthesis under heterotrophic conditions,which provides a new sight in improving oil content of A.protothecoides by means of genetic engineering,and lays a foundation for future study of miRNA in A.protothecoides.