Transcriptome And Proteome Analysis And The Prediction Of Oil Biosynthesis Pathway In Dunaliella Parva

Microalgal lipid is a promising feedstock for biodiesel production,but the high cost seriously restricts the development of microalgal lipid.Oil content is one of the important factors which determine the cost of biodiesel production.The biomass of microalgae is higher,but the oil content is lower under the condition of sufficient nitrogen source.It is an important problem to increase the oil content of microalgae under the condition of sufficient nitrogen source up to now.D.parva is a unicellular green alga.It can accumulate lipids and large amounts of carotenoids,and adapt to abiotic stresses such as high-salt.In addition,it lacks cell wall,which is in favour of genetic manipulation and easier product extraction.These advantages made D.parva appealing compared with model microalgae as feedstock of biodiesel production.Our former study found that nitrogen limitation significantly induced the increase of oil content in D.parva,but the biomass obviously decreased.The regulatory mechanism was not clear.This paper studied the change of transcriptome and proteome under the condition of nitrogen limitation in order to solve the problem of opposite changing trend between the biomass and lipid content in D.parva,which was helpful for us to identify key enzyme genes and regulatory genes in oil biosynthesis pathway.In addition,it would provide the basis for simultaneous increase of biomass and lipid content by manipulating regulatory genes.Culture conditions were optimized by single factor experiment in D.parva.The single factor experiment showed that sodium nitrate and sodium chloride had significant influence on the growth of D.parva in the certain concentrations.However,the other factors such as sodium bicarbonate,potassium chloride,magnesium sulfate,calcium chloride,ferric citrate,A₅ solution and pH value,had a little influence on the growth of D.parva.The influence of sodium nitrate concentrations on the growth of D.parva would provide the basis for the selection of nitrogen sufficient and nitrogen limited conditions.The de novo transcriptome sequencing of D.parva was performed by Illumina Hiseq2000 platform in control?nitrogen sufficient condition,5.0mM sodium nitrate,SCH-5.0mMA?and treated sample?nitrogen limitation condition,0.5mM sodium nitrate,SCH-0.5mMA?.A total 29380572 and 29815432 raw reads were generated in control and treated sample,respectively.Filtration resulted in 26304060 and 26797446 clean reads in control and treated sample,respectively.Clean reads of two samples were assembled using Trinity software.The assembly produced 41357,40015 and 35213 unigenes in control,treated sample and All-unigene,respectively.The proportions of unigenes with length of more than 1000bp,are 23.08%,28.17%and 36.01%in control,treated sample and All-unigene,respectively.In addtion,the N50 values are 1044bp,1173bp and 1346bp in control,treated sample and All-unigene,respectively.These results indicated that our study obtained better assembly quality,which provided foundation for gene annotation and pathway analysis in the future.Length distribution of CDS shows that 24378 unigenes?93.27%of the total unigenes?have CDS with length of more than 300bp,which indicates better assembly quality.35213 unigenes were obtained through assembly.The average length of unigenes is 916bp.The N50 value is 1346bp.All unigenes were aligned against databases such as Nr,Nt,Swiss-Prot,COG,GO and KEGG,and 21568 unigenes were annotated.More unigenes were identified in primary metabolic pathways such as carbohydrate metabolism,energy metabolism,amino acid metabolism and nucleotide metabolism,which resulted in complete metabolic pathways.Fewer unigenes were identified in secondary metabolic pathways because of lower level of gene expression,which resulted in relatively incomplete metabolic pathways.A more comprehensive understanding of the potential function of unigenes could be obtained using three classification methods?COG,GO and KEGG?in D.parva,which provided foundation for studying specific target genes in the future.The differentially expressed genes were analysed between control and treated sample in D.parva.There were 1529 significant differentially expressed genes between control and treated sample.The GO and KEGG classification of differentially expressed genes showed that there were significant differences in metabolic pathways such as photosynthesis,nitrogen metabolism,amino sugar and nucleotide sugar metabolism,RNA transport,starch and sucrose metabolism,amino acid metabolism,glycerophospholipid metabolism,protein processing,purine and pyrimidine metabolism.The products of differentially expressed genes were mainly located in plastid,chloroplast and thylakoid.The obtainment of differentially expressed genes provided foundation for studying the response mechanism of oil biosynthesis to nitrogen limitation in D.parva.The differentially expressed proteins were analysed between control and treated sample in D.parva.There were 386 significant differentially expressed proteins between control and treated sample.The GO and KEGG classification of differentially expressed proteins showed that there were significant differences in metabolic pathways such as photosynthesis,TCA cycle,oxidative phosphorylation,pyruvate metabolism,biosynthesis of secondary metabolites,nitrogen metabolism,starch and sucrose metabolism,glycolysis and gluconeogenesis,fatty acid biosynthesis and amino acid metabolism.The differentially expressed proteins were mainly located in cytoplasm part,plastid and chloroplast.A comprehensive analysis of differentially expressed genes and proteins was helpful for us to deeply understand the response mechanism of oil biosynthesis to nitrogen limitation in D.parva.Based on KEGG annotation,we constructed fatty acid biosynthesis pathway,triacylglycerol biosynthesis pathway and starch metabolism pathway in D.parva.In addtion,the expression levels of related genes in three metabolic pathways were analysed.A regulatory gene of oil biosynthesis?wri1?and a Lip gene related to oil catabolism were identified among differentially expressed genes.The full-length cDNAs of wri1 and Lip were obtained by RACE method.At the same time,their promoters were obtained using Genome Walking method.The expression levels of wri1 and Lip were analysed between control and treated sample by real-time PCR.The results showed that nitrogen limitation could significantly induce the expression of these two genes.Perhaps these two genes were closely related to oil accumulation.Besides,we purified protein wri1 and prepared its antibody.Then the chromatin immunoprecipitation experiment was performed using the antibody of wri1 and the D.parva samples in order to determine the gene fragments combined with protein wri1,and understand the target genes regulated by wri1.These results provided foundation for subsequent researches.This paper studied the changes of transcriptome and proteome between control?nitrogen sufficient condition?and treated sample?nitrogen limitation condition?in D.parva,constructed oil biosynthesis pathway,and identified an important regulatory gene of oil biosynthesis?wri1?.In addtion,we described regulatory gene wri1 and Lip gene related to oil catabolism.We obtained the genes of the biosynthesis pathway of oil and starch,and identified an important transcription factor gene wri1 related to the regulation of oil biosynthesis through the analysis of the changes in transcriptome and proteome under nitrogen limitation condition.In addition,we found that nitrogen limitation could significantly induce the expression of wri1 and Lip.We obtained the enriched DNA by transcription factor wri1 through prokaryotic expression,affinity purification,the preparation of antibody and chromatin immunoprecipitation.The enriched DNA will be sequenced in the future.These works provided foundation for illuminating the regulatory mechanism of lipid biosynthesis and modifying D.parva in the future.