BACKGROUND
the demand of energy is increasing as the world population and global economy continue to grow.
microalgae-based biodiesel, which can realize carbon-neutral by photosynthetic carbon fixation via the microalgae’s growth  <cit> , is a renewable and sustainable energy source.
chlorella, one of eukaryotic, unicellular and photosynthetic microorganism, widely distributes in freshwater environment and is capable of accumulating excess lipid in nitrogen-limited condition.
moreover, chlorella were used as a model system for investigating photosynthetic carbon fixation  <cit> .
due to its various and robust metabolic capacities, chlorella has received increasingly attention as promising microalgae to produce biomass  <cit> , biodiesel  <cit>  as well as high additional-value products  <cit> .
currently, chlorella is one of the best microalgae as oil feedstock for the production of biodiesel  <cit> .
particularly, in nitrogen-limited condition, chlorella can alter the metabolic pathways to accumulate a high proportion of lipid which can be used for biodiesel production .
although the metabolic transition has been identified in the lipid accumulation process  <cit> , many global changes remain poorly understood, such as genomic information, differential genes expression.
as a consequence, the lipid production from naturally occurring chlorella strains is much lower than the theoretical maximum  <cit> , making the cost of biodiesel prohibitively high  <cit> .
one primary cause is the limited understanding of the metabolic pathways in microalgae regulating the lipid metabolism in general and lipid biosynthesis and accumulation in particular  <cit> .
another cause is the lack of genomic information of some oleaginous but non-model microalgae, which largely hampers the investigation of the transcribed genes and genetic modification in these microalgae to accumulate lipid and other valuable products .
transcriptome sequencing could be an efficient and relatively economical method to obtain the functional genomic information without the genomic information  <cit> , providing an initial, broad view of lipid accumulation in nitrogen-limited condition  <cit>  and photosynthetic carbon fixation.
a growing number of transcriptomes of oleaginous microalgae were de novo sequenced, assembled and annotated to investigate the regulatory mechanism of lipid accumulation .
in our previous work, we have already identified the metabolic pathways related to lipid accumulation in c. sorokiniana based on two transcriptome datasets  <cit> .
in this present study, we sequenced another four transcriptome datasets and analyzed all six transcriptome datasets together to elucidate differential gene expression involved in the lipid accumulation and photosynthetic carbon fixation.
in our experiments, the quantity of lipid accumulated under nitrogen-limited condition can be  <dig>  times than that under nitrogen-replete condition, making c. sorokiniana a promising microalgae strain to produce biodiesel.
then all the six transcriptome datasets were de novo assembled, annotated together, and differential genes expression was analyzed as well.
finally, rt-qpcr was conducted for  <dig> genes involving in the lipid accumulation and photosynthetic carbon fixation.
our results provide an insight into the regulation of lipid metabolism and photosynthetic carbon fixation in c. sorokiniana at the transcriptomic level and may contribute to genetic modification in microalgae to improve lipid productivity and carbon fixation efficiency.
RESULTS
biomass and lipid content under different cultivation conditions
six different cultivation conditions were selected to culture c. sorokiniana , and the growth and lipid content profiles under these conditions were shown in fig.
<dig>  with 4 % glucose as carbon source, the optical density of the culture at 680 nm  in nitrogen-limited condition  was almost equal to that in nitrogen-replete condition  before 48 h. after 48 h the od <dig> in nitrogen-replete condition began to become higher than that in nitrogen-limited condition .
the higher od <dig> resulted in the more glucose consumption  for cell growth, not for lipid production.
the nitrogen-limited condition could induce c. sorokiniana to accumulate more lipid.
after 48 h, the fluorescence intensity of lipid dyed with nile red in nitrogen-limited condition was higher than that in nitrogen-replete condition, meaning that the cells in nitrogen-limited condition accumulated more lipid.
at 84 h, the fluorescence intensity in nitrogen-limited condition was as high as  <dig>  times than that in nitrogen-replete condition .table  <dig> the general information for each sample
kno3
od680
fluorescence intensityd
athe transcriptome datasets of these samples were sequenced before
broom temperature
cdo not take the influence of light into account
dthe fluorescence intensity of lipid dyed with nile red
fig.
<dig> growth and lipid content of c. sorokiniana under different conditions.
a: growth and lipid content of c. sorokiniana under nitrogen-limited and nitrogen-replete conditions.
b: the consumption of glucose under nitrogen-limited and nitrogen-replete conditions; c: growth and lipid content of c. sorokiniana in light; d: growth and lipid content of c. sorokiniana in dark
when c. sorokiniana was cultivated in light, the fluorescence intensity increased steadily from  <dig>  ±  <dig>  at third day to  <dig>  ±  <dig>  at eighth day, increasing by  <dig>  times and the od <dig> increased continuously from  <dig>  ±  <dig>  to  <dig>  ±  <dig>  over the whole cultivation period .
these indicated that c. sorokiniana could absorb co <dig> as carbon source to reproduce and also transform it into lipid accumulated in cells, which provided a promising strategy to alleviate global warming and energy crisis.
when cultured heterotrophically in darkness with nitrogen-limited condition, c. sorokiniana accumulated lipid as well and the fluorescence intensity increased by  <dig>  times at 84 h .
compared with photoautotrophy, heterotrophy could make c. sorokiniana yield more biomass and achieve higher lipid productivity .
sequencing and de novo assembly
after illumina hiseq <dig> paired-end sequencing, over  <dig> , <dig> raw reads were generated and are available at the ncbi sra database .
all the raw reads were subjected to trimming based on base quality score and read length, and  <dig> , <dig> clean reads were generated , which were de novo assembled into  <dig>  contigs with n <dig> of  <dig>  bp.
after clustered,  <dig>  non-redundant contigs, ranging from 200 bp to  <dig>  bp, were generated with an average length of  <dig>  bp , which was used for the following analysis.
the transcriptome short assembly project has been deposited at ddbj/embl/genbank under the accession gapd <dig>  the version described in this paper is the first version gapd <dig> fig.
<dig> characteristics of homology search of assembly against the nr database.
a: the length distribution of contigs assembled.
b: the length distribution of contigs with match  in nr database.
c: species distribution of the total homologous sequences with e-value ≤  <dig> e- <dig>  d: e-value distribution of blastx hits for each contigs with e-value ≤  <dig> e- <dig>  we used the first hit of each search results for analysis
annotation of contigs
after compared against the ncbi’s nr database using blastx,  <dig>  contigs  were found having homologous sequence in nr database .
due to the lack of genome information, a large proportion of the contigs  could not be matched to homologous sequence in any database, among which  <dig>  potential coding regions were predicted using transdecoder .
these predicted coding regions seem to be new genes, and their functions should be further confirmed.
ec number and ko identifier were also assigned from the annotation results of kegg, and  <dig>  contigs were assigned with ec number .
there were  <dig>  contigs which were all matched with homologous sequences in all the databases used .
particularly, the length of most contigs with homologous sequence in nr database were between  <dig> and 2500 bp  and the match efficiency decreased as the length of contigs increased , indicating that most genes of c. sorokiniana were in the range of 500 bp and 2500 bp.
moreover, the homologous sequences matched in nr came from closely related green microalgae species, including c. variabilis , coccomyxa subellipsoidea c- <dig>  and volvox carteri f. nagariensis  , based on which we selected chlorella sp.
nc64a as the candidate for predicting transcription factors.
similar results were also reported in the transcriptomic analysis of dunaliella tertiolecta  <cit>  and chlamydomonas moewusii  <cit> .
the e-value distribution of the top match in nr database showed that  <dig>  % of the matched sequences had e-value ≤  <dig> e- <dig>  and  <dig>  % ranged from  <dig> e- <dig> to  <dig> e- <dig> .
similar results were also reported in the de novo transcriptomic analysis of ambystoma mexicanum  <cit> .fig.
<dig> venn diagram of annotated contigs’ number in different database.
nr: ncbi’s non-redundant database.
cog: clusters of orthologous groups  database.
ec number and ko: the annotation results of kegg database.
each figure means the number of annotated contigs in corresponding databases
function classification and transcription factor analysis
<dig>  contigs were assigned with at least  <dig> cog category and  <dig> contigs had no homologous sequence in nr database but matched homologous sequence in cog database .
among the  <dig> cog categories, the cluster for “general function prediction only” accounted for the largest group , followed by “replication, recombination and repair”  and “transcription” .
in addition, the following categories contained the minimum number of contigs: “nuclear structure” , “cell motility” , “rna processing and modification” .
we even did not find any contig assigned to the category “extracellular structures” .fig.
<dig> histogram presentation of clusters of orthologous groups  categories.
out of  <dig> nr hits,  <dig> contigs were assigned at least one cog category among the  <dig> categories
transcription factors, most of which were protein, also play significant roles in responding to environment stress by regulating gene expression which were classified into different families such as sbp, c3h and bhlh.
after searching against the transcription factors database of chlorella sp.
nc64a  <cit> ,  <dig> contigs were found having homologous sequences and assigned into  <dig> families .
the most abundant transcription factor family was sbp family related to flower development in plant  <cit> .
c3h family was the next abundant transcription factor family, which played an important role during arabidopsis embryogenesis and functioned primarily in the apical domain of the embryo  <cit>  .
in terms of the specific function of these transcription factor families in c. sorokiniana, further studies should be further conducted.
moreover, the differential expression of transcription factor were also investigated.
<dig> contigs assigned to  <dig> different transcription factors were found at least 2-fold up-regulated while only  <dig> contigs homologous with  <dig> different transcription factors at least 2-fold down-regulated in nitrogen-limited condition.
when c. sorokiniana was cultivated in light, we found  <dig> contigs homologous with  <dig> transcription factors at least 2-fold up-regulated and  <dig> contigs annotated as  <dig> transcription factors at least 2-fold down-regulated .table  <dig> transcription factor families identified in c. sorokiniana
atranscription factor id.
in the transcription factor database , each transcriptome factor family contains several items and each item assigned with a tf id
*means at least 2-fold up-regulation
#means at least 2-fold down-regulation
up to now, it have been proved that dof-type transcription factor and bhlh family have the function of regulating lipid accumulation in plants .
in this study, two transcription factors  in bhlh family were identified and found both up-regulated in nitrogen-limited condition, which further confirm the significance of bhlh family in the in lipid accumulation .
at the same time, others transcription factors assigned to other families were also found with at least 2-fold expression change in respond to nitrogen deprivation , and most of these transcription factor families were also reported to be up-regulated in chlamydomonas reinhardtii cultivated in n-deprived condition especially the myb-related, sbp and c3h family  <cit> .
thus, regulating these transcription factors would be a potential approach to increase the lipid accumulation  <cit> .
moreover, many transcription factors related to photosynthetic carbon fixation were also found to be up-regulated or down-regulated in light .
these results would be very useful for the improvement of photosynthetic carbon assimilation in microalgae as few transcription factors involving in photosynthetic carbon assimilation were investigated .
genes expression quantification
the expression abundance of genes obtained from the annotation of assembled contigs were quantified using fpkm method  <cit> .
when comparing genes expression abundance of sample a  with that of sample c , we found  <dig> genes were at least 2-fold up-regulated and  <dig> genes were at least 2-fold down-regulated in nitrogen-limited condition .
then comparing genes expression abundance between sample b  and sample d ,  <dig> genes were found at least 2-fold up-regulated, while  <dig> genes down-regulated in sample b .fig.
<dig> differential genes expression in c. sorokiniana of six cultivation conditions.
a: at least 2-fold up-regulation in nitrogen-limited condition.
b: at least 2-fold down-regulation in nitrogen-limited condition.
c: no difference between nitrogen-limited and nitrogen-replete condition .
d: differential genes expression between light and dark condition
we also investigated the gene expression profiles for cultivation with 48 h and 84 h, and found  <dig> genes at least 2-fold up-regulated in nitrogen-limited condition at both cultivation times , while  <dig> genes at least 2-fold down-regulated .
the expression change of most genes  were less than 2-fold, and  <dig> genes were found identical at both time .
interestingly, we found more genes with at least 2-fold up-regulation and less genes with at least 2-fold down-regulation at 84 h compared with the counterparts at 48 h .
the reason for this may be the concentration of nitrogen in the media declined with c. sorokiniana growing, which could induce more genes to increase its transcriptional level as a response to the lower concentration of nitrogen.
to investigate the differential expression of genes related to lipid accumulation, we mainly focused on the lipid-related metabolic pathways, including fatty acid biosynthesis and catabolism pathway, triacylglycerol biosynthesis pathway, and starch biosynthesis and catabolism pathway .
in these metabolic pathways, we found most genes up-regulated in nitrogen-limited condition at both 48 h and 84 h except those in starch metabolic pathway.
on the contrary, the starch biosynthesis pathway was down-regulated in nitrogen-limited condition, while the starch catabolic pathway was up-regulated, which was also reported in other microalgae  <cit> , indicating microalgae preferred to synthesis lipid rather than starch in nitrogen-limited condition.table  <dig> differential gene expression in lipid accumulation related pathways
a
log2fc=log2fpkmnitrogen−limitedfpkmnitrogen−replete
when investigating differential gene expression between light and dark condition, we found  <dig> genes with expression change more than 2-fold, comprising  <dig> genes up-regulated in light and  <dig> genes up-regulated in dark.
similarly, the expression change of most genes  were less than 2-fold .
in terms of photosynthetic carbon fixation, we particularly focused on the genes involving in calvin cycle and found all genes except tpi  up-regulated in light.
the pgk , rbcl  and rpk  even found up-regulated by  <dig> ,  <dig>  and  <dig>  times, respectively .
unfortunately, there were still many genes with differential expression annotated as “hypothetical protein with unknown functions” in the annotation results .
therefore, it will be necessary to investigate the potential functions of these genes.table  <dig> differential gene expression in calvin cycle
a
log2fc=log2fpkmlightfpkmdark
real-time quantitative pcr analysis
<dig> genes were selected to perform real-time quantitative pcr .
in the lipid metabolic pathways ,  <dig> genes  showed up-regulation in nitrogen-limited condition, especially bc and kar.
however,  <dig> genes  were found down-regulated in the nitrogen-limited condition.
the down-regulation of acc and up-regulation of bc under nitrogen-limited condition were also reported in neochloris oleoabundans  <cit> .fig.
<dig> the differentially expressed genes profiles detected by rt-qpcr.
c. sorokiniana was cultivated under six different conditions.
a: genes involving in lipid accumulation; b: genes involving in calvin cycle.
bc: biotin carboxylase; kas ii: 3-oxoacyl- synthase ii; kas iii: 3-oxoacyl- synthase iii; kar: beta-ketoacyl-acp reductase; agpat: 1-acyl-sn-glycerol-3-phosphate acyltransferase; dgat: diacylglycerol o-acyltransferase; acc: acetyl-coa carboxylase; mat: malonyltransferase; ss: starch synthase; be:  <dig> -α-glucan branching enzyme; aox: acyl-coa oxidase; gpat: glycerol-3-phosphate o-acyltransferase; rbcl: ribulose-bisphosphate carboxylase large chain; sebp: sedoheptulose-bisphosphatase; rpe: ribulose-phosphate 3-epimerase; aldo: fructose-bisphosphate aldolase.
standard error of mean for three technical replicates is represented by the error bars
moreover, we found the down-regulation of starch biosynthesis pathway  in nitrogen-limited condition, indicating that the starch biosynthesis pathway might be inhibited .
this result was also reported in n. oleoabundans  <cit> .
<dig> genes involving in carbon fixation pathway  were found all up-regulated in light .
among the  <dig> genes with rt-qpcr analysis,  <dig> genes showed the similar expression patterns as those identified by the transcriptomic analysis.
only the gene coding starch synthase showed inconsistent result in term of differential expression between the rt-qpcr analysis and transcriptomic analysis at 84 h. based on the result of rt-qpcr analysis, this gene was found down-regulated in nitrogen-limited condition at 84 h, while the transcriptomic analysis result showed it was up-regulated in the corresponding condition.
CONCLUSIONS
this study not only provided transcriptome datasets of c. sorokiniana under six different conditions, but also new biological insights into the expression of genes associated with lipid accumulation and photosynthetic carbon fixation.
based on our study, it is clear that the application of this approach can contribute to the generation of interesting hypotheses for both fundamental and applied research.
moreover, the c. sorokiniana’s transcriptome data could be a contribution for elucidating the physiology and evolution of the chlorophyceans.

 the demand of energy is increasing as the world population and global economy continue to grow. microalgae-based biodiesel, which can realize carbon-neutral by photosynthetic carbon fixation via the microalgae’s growth, is a renewable and sustainable energy source. chlorella, one of eukaryotic, unicellular and photosynthetic microorganism, widely distributes in freshwater environment and is capable of accumulating excess lipid in nitrogen-limited condition. moreover, chlorella were used as a model system for investigating photosynthetic carbon fixation. due to its various and robust metabolic capacities, chlorella has received increasingly attention as promising microalgae to produce biomass, biodiesel as well as high additional-value products. currently, chlorella is one of the best microalgae as oil feedstock for the production of biodiesel. particularly, in nitrogen-limited condition, chlorella can alter the metabolic pathways to accumulate a high proportion of lipid which can be used for biodiesel production. although the metabolic transition has been identified in the lipid accumulation process, many global changes remain poorly understood, such as genomic information, differential genes expression. as a consequence, the lipid production from naturally occurring chlorella strains is much lower than the theoretical maximum, making the cost of biodiesel prohibitively high. one primary cause is the limited understanding of the metabolic pathways in microalgae regulating the lipid metabolism in general and lipid biosynthesis and accumulation in particular. another cause is the lack of genomic information of some oleaginous but non-model microalgae, which largely hampers the investigation of the transcribed genes and genetic modification in these microalgae to accumulate lipid and other valuable products. transcriptome sequencing could be an efficient and relatively economical method to obtain the functional genomic information without the genomic information, providing an initial, broad view of lipid accumulation in nitrogen-limited condition and photosynthetic carbon fixation. a growing number of transcriptomes of oleaginous microalgae were de novo sequenced, assembled and annotated to investigate the regulatory mechanism of lipid accumulation. in our previous work, we have already identified the metabolic pathways related to lipid accumulation in c. sorokiniana based on two transcriptome datasets. in this present study, we sequenced another four transcriptome datasets and analyzed all six transcriptome datasets together to elucidate differential gene expression involved in the lipid accumulation and photosynthetic carbon fixation. in our experiments, the quantity of lipid accumulated under nitrogen-limited condition can be times than that under nitrogen-replete condition, making c. sorokiniana a promising microalgae strain to produce biodiesel. then all the six transcriptome datasets were de novo assembled, annotated together, and differential genes expression was analyzed as well. finally, rt-qpcr was conducted for genes involving in the lipid accumulation and photosynthetic carbon fixation. our results provide an insight into the regulation of lipid metabolism and photosynthetic carbon fixation in c. sorokiniana at the transcriptomic level and may contribute to genetic modification in microalgae to improve lipid productivity and carbon fixation efficiency.  biomass and lipid content under different cultivation conditions six different cultivation conditions were selected to culture c. sorokiniana, and the growth and lipid content profiles under these conditions were shown in fig.  with 4 % glucose as carbon source, the optical density of the culture at 680 nm in nitrogen-limited condition was almost equal to that in nitrogen-replete condition before 48 h. after 48 h the od in nitrogen-replete condition began to become higher than that in nitrogen-limited condition. the higher od resulted in the more glucose consumption for cell growth, not for lipid production. the nitrogen-limited condition could induce c. sorokiniana to accumulate more lipid. after 48 h, the fluorescence intensity of lipid dyed with nile red in nitrogen-limited condition was higher than that in nitrogen-replete condition, meaning that the cells in nitrogen-limited condition accumulated more lipid. at 84 h, the fluorescence intensity in nitrogen-limited condition was as high as times than that in nitrogen-replete condition.table the general information for each sample kno3 od680 fluorescence intensityd athe transcriptome datasets of these samples were sequenced before broom temperature cdo not take the influence of light into account dthe fluorescence intensity of lipid dyed with nile red fig. growth and lipid content of c. sorokiniana under different conditions. a: growth and lipid content of c. sorokiniana under nitrogen-limited and nitrogen-replete conditions. b: the consumption of glucose under nitrogen-limited and nitrogen-replete conditions; c: growth and lipid content of c. sorokiniana in light; d: growth and lipid content of c. sorokiniana in dark when c. sorokiniana was cultivated in light, the fluorescence intensity increased steadily from  ±  at third day to  ±  at eighth day, increasing by times and the od increased continuously from  ±  to  ±  over the whole cultivation period. these indicated that c. sorokiniana could absorb co as carbon source to reproduce and also transform it into lipid accumulated in cells, which provided a promising strategy to alleviate global warming and energy crisis. when cultured heterotrophically in darkness with nitrogen-limited condition, c. sorokiniana accumulated lipid as well and the fluorescence intensity increased by times at 84 h. compared with photoautotrophy, heterotrophy could make c. sorokiniana yield more biomass and achieve higher lipid productivity. sequencing and de novo assembly after illumina hiseq paired-end sequencing, over, raw reads were generated and are available at the ncbi sra database. all the raw reads were subjected to trimming based on base quality score and read length, and, clean reads were generated, which were de novo assembled into contigs with n of  bp. after clustered, non-redundant contigs, ranging from 200 bp to  bp, were generated with an average length of  bp, which was used for the following analysis. the transcriptome short assembly project has been deposited at ddbj/embl/genbank under the accession gapd the version described in this paper is the first version gapd fig. characteristics of homology search of assembly against the nr database. a: the length distribution of contigs assembled. b: the length distribution of contigs with match in nr database. c: species distribution of the total homologous sequences with e-value ≤  e- d: e-value distribution of blastx hits for each contigs with e-value ≤  e- we used the first hit of each search results for analysis annotation of contigs after compared against the ncbi’s nr database using blastx, contigs were found having homologous sequence in nr database. due to the lack of genome information, a large proportion of the contigs could not be matched to homologous sequence in any database, among which potential coding regions were predicted using transdecoder. these predicted coding regions seem to be new genes, and their functions should be further confirmed. ec number and ko identifier were also assigned from the annotation results of kegg, and contigs were assigned with ec number. there were contigs which were all matched with homologous sequences in all the databases used. particularly, the length of most contigs with homologous sequence in nr database were between and 2500 bp and the match efficiency decreased as the length of contigs increased, indicating that most genes of c. sorokiniana were in the range of 500 bp and 2500 bp. moreover, the homologous sequences matched in nr came from closely related green microalgae species, including c. variabilis, coccomyxa subellipsoidea c- and volvox carteri f. nagariensis, based on which we selected chlorella sp. nc64a as the candidate for predicting transcription factors. similar results were also reported in the transcriptomic analysis of dunaliella tertiolecta and chlamydomonas moewusii. the e-value distribution of the top match in nr database showed that  % of the matched sequences had e-value ≤  e- and  % ranged from e- to e-. similar results were also reported in the de novo transcriptomic analysis of ambystoma mexicanum.fig. venn diagram of annotated contigs’ number in different database. nr: ncbi’s non-redundant database. cog: clusters of orthologous groups database. ec number and ko: the annotation results of kegg database. each figure means the number of annotated contigs in corresponding databases function classification and transcription factor analysis contigs were assigned with at least cog category and contigs had no homologous sequence in nr database but matched homologous sequence in cog database. among the cog categories, the cluster for “general function prediction only” accounted for the largest group, followed by “replication, recombination and repair” and “transcription”. in addition, the following categories contained the minimum number of contigs: “nuclear structure”, “cell motility”, “rna processing and modification”. we even did not find any contig assigned to the category “extracellular structures”.fig. histogram presentation of clusters of orthologous groups categories. out of nr hits, contigs were assigned at least one cog category among the categories transcription factors, most of which were protein, also play significant roles in responding to environment stress by regulating gene expression which were classified into different families such as sbp, c3h and bhlh. after searching against the transcription factors database of chlorella sp. nc64a, contigs were found having homologous sequences and assigned into families. the most abundant transcription factor family was sbp family related to flower development in plant. c3h family was the next abundant transcription factor family, which played an important role during arabidopsis embryogenesis and functioned primarily in the apical domain of the embryo. in terms of the specific function of these transcription factor families in c. sorokiniana, further studies should be further conducted. moreover, the differential expression of transcription factor were also investigated. contigs assigned to different transcription factors were found at least 2-fold up-regulated while only contigs homologous with different transcription factors at least 2-fold down-regulated in nitrogen-limited condition. when c. sorokiniana was cultivated in light, we found contigs homologous with transcription factors at least 2-fold up-regulated and contigs annotated as transcription factors at least 2-fold down-regulated.table transcription factor families identified in c. sorokiniana atranscription factor id. in the transcription factor database, each transcriptome factor family contains several items and each item assigned with a tf id *means at least 2-fold up-regulation #means at least 2-fold down-regulation up to now, it have been proved that dof-type transcription factor and bhlh family have the function of regulating lipid accumulation in plants. in this study, two transcription factors in bhlh family were identified and found both up-regulated in nitrogen-limited condition, which further confirm the significance of bhlh family in the in lipid accumulation. at the same time, others transcription factors assigned to other families were also found with at least 2-fold expression change in respond to nitrogen deprivation, and most of these transcription factor families were also reported to be up-regulated in chlamydomonas reinhardtii cultivated in n-deprived condition especially the myb-related, sbp and c3h family. thus, regulating these transcription factors would be a potential approach to increase the lipid accumulation. moreover, many transcription factors related to photosynthetic carbon fixation were also found to be up-regulated or down-regulated in light. these results would be very useful for the improvement of photosynthetic carbon assimilation in microalgae as few transcription factors involving in photosynthetic carbon assimilation were investigated. genes expression quantification the expression abundance of genes obtained from the annotation of assembled contigs were quantified using fpkm method. when comparing genes expression abundance of sample a with that of sample c, we found genes were at least 2-fold up-regulated and genes were at least 2-fold down-regulated in nitrogen-limited condition. then comparing genes expression abundance between sample b and sample d, genes were found at least 2-fold up-regulated, while genes down-regulated in sample b.fig. differential genes expression in c. sorokiniana of six cultivation conditions. a: at least 2-fold up-regulation in nitrogen-limited condition. b: at least 2-fold down-regulation in nitrogen-limited condition. c: no difference between nitrogen-limited and nitrogen-replete condition. d: differential genes expression between light and dark condition we also investigated the gene expression profiles for cultivation with 48 h and 84 h, and found genes at least 2-fold up-regulated in nitrogen-limited condition at both cultivation times, while genes at least 2-fold down-regulated. the expression change of most genes were less than 2-fold, and genes were found identical at both time. interestingly, we found more genes with at least 2-fold up-regulation and less genes with at least 2-fold down-regulation at 84 h compared with the counterparts at 48 h. the reason for this may be the concentration of nitrogen in the media declined with c. sorokiniana growing, which could induce more genes to increase its transcriptional level as a response to the lower concentration of nitrogen. to investigate the differential expression of genes related to lipid accumulation, we mainly focused on the lipid-related metabolic pathways, including fatty acid biosynthesis and catabolism pathway, triacylglycerol biosynthesis pathway, and starch biosynthesis and catabolism pathway. in these metabolic pathways, we found most genes up-regulated in nitrogen-limited condition at both 48 h and 84 h except those in starch metabolic pathway. on the contrary, the starch biosynthesis pathway was down-regulated in nitrogen-limited condition, while the starch catabolic pathway was up-regulated, which was also reported in other microalgae, indicating microalgae preferred to synthesis lipid rather than starch in nitrogen-limited condition.table differential gene expression in lipid accumulation related pathways a log2fc=log2fpkmnitrogen−limitedfpkmnitrogen−replete when investigating differential gene expression between light and dark condition, we found genes with expression change more than 2-fold, comprising genes up-regulated in light and genes up-regulated in dark. similarly, the expression change of most genes were less than 2-fold. in terms of photosynthetic carbon fixation, we particularly focused on the genes involving in calvin cycle and found all genes except tpi up-regulated in light. the pgk, rbcl and rpk even found up-regulated by, and times, respectively. unfortunately, there were still many genes with differential expression annotated as “hypothetical protein with unknown functions” in the annotation results. therefore, it will be necessary to investigate the potential functions of these genes.table differential gene expression in calvin cycle a log2fc=log2fpkmlightfpkmdark real-time quantitative pcr analysis genes were selected to perform real-time quantitative pcr. in the lipid metabolic pathways, genes showed up-regulation in nitrogen-limited condition, especially bc and kar. however, genes were found down-regulated in the nitrogen-limited condition. the down-regulation of acc and up-regulation of bc under nitrogen-limited condition were also reported in neochloris oleoabundans.fig. the differentially expressed genes profiles detected by rt-qpcr. c. sorokiniana was cultivated under six different conditions. a: genes involving in lipid accumulation; b: genes involving in calvin cycle. bc: biotin carboxylase; kas ii: 3-oxoacyl- synthase ii; kas iii: 3-oxoacyl- synthase iii; kar: beta-ketoacyl-acp reductase; agpat: 1-acyl-sn-glycerol-3-phosphate acyltransferase; dgat: diacylglycerol o-acyltransferase; acc: acetyl-coa carboxylase; mat: malonyltransferase; ss: starch synthase; be: -α-glucan branching enzyme; aox: acyl-coa oxidase; gpat: glycerol-3-phosphate o-acyltransferase; rbcl: ribulose-bisphosphate carboxylase large chain; sebp: sedoheptulose-bisphosphatase; rpe: ribulose-phosphate 3-epimerase; aldo: fructose-bisphosphate aldolase. standard error of mean for three technical replicates is represented by the error bars moreover, we found the down-regulation of starch biosynthesis pathway in nitrogen-limited condition, indicating that the starch biosynthesis pathway might be inhibited. this result was also reported in n. oleoabundans. genes involving in carbon fixation pathway were found all up-regulated in light. among the genes with rt-qpcr analysis, genes showed the similar expression patterns as those identified by the transcriptomic analysis. only the gene coding starch synthase showed inconsistent result in term of differential expression between the rt-qpcr analysis and transcriptomic analysis at 84 h. based on the result of rt-qpcr analysis, this gene was found down-regulated in nitrogen-limited condition at 84 h, while the transcriptomic analysis result showed it was up-regulated in the corresponding condition.  this study not only provided transcriptome datasets of c. sorokiniana under six different conditions, but also new biological insights into the expression of genes associated with lipid accumulation and photosynthetic carbon fixation. based on our study, it is clear that the application of this approach can contribute to the generation of interesting hypotheses for both fundamental and applied research. moreover, the c. sorokiniana’s transcriptome data could be a contribution for elucidating the physiology and evolution of the chlorophyceans. 
six cultivation conditions were selected to cultivate c. sorokiniana. the paucity of genomic information, however, limits the development of genome-based genetic modification to improve lipid production in many microalgae. the genes with expression change under different conditions are highly likely the potential targets for genetic modification to improve lipid production and co fixation efficiency in oleaginous microalgae. finally, genes were selected to conduct rt-qpcr and genes showed the similar results as those identified by transcriptomic analysis in term of differential expression. when cultivated in light with nitrogen-limited supply, c. sorokiniana can use carbon dioxide to accumulate lipid. here, we describe the de novo sequencing, transcriptome assembly, annotation and differential expression analysis for chlorella sorokiniana cultivated in different conditions to reveal the change of genes expression associated with lipid accumulation and photosynthetic carbon fixation. lipid content of c. sorokiniana under nitrogen-limited condition was times than that under nitrogen-replete condition. de novo transcriptomic analyses have generated enormous information over c. sorokiniana, revealing a broad overview of genomic information related to lipid accumulation and photosynthetic carbon fixation. chlorella sorokinianatranscriptomelipid accumulationphotosynthetic carbon fixationrna-seqnational basic research program of china 2011cb200900wang qinhong issue-copyright-statement© the author 2016 after preprocessed, these reads were de novo assembled into contigs among which contigs were found homologous sequences in public databases through blastx. then, transcriptome of c. sorokiniana was sequenced using illumina paired-end sequencing technology, and, raw reads with length of 100 bp were produced.
 microalgae, which can absorb carbon dioxide and then transform it into lipid, are promising candidates to produce renewable energy, especially biodiesel. the paucity of genomic information, however, limits the development of genome-based genetic modification to improve lipid production in many microalgae. here, we describe the de novo sequencing, transcriptome assembly, annotation and differential expression analysis for chlorella sorokiniana cultivated in different conditions to reveal the change of genes expression associated with lipid accumulation and photosynthetic carbon fixation.  six cultivation conditions were selected to cultivate c. sorokiniana. lipid content of c. sorokiniana under nitrogen-limited condition was times than that under nitrogen-replete condition. when cultivated in light with nitrogen-limited supply, c. sorokiniana can use carbon dioxide to accumulate lipid. then, transcriptome of c. sorokiniana was sequenced using illumina paired-end sequencing technology, and, raw reads with length of 100 bp were produced. after preprocessed, these reads were de novo assembled into contigs among which contigs were found homologous sequences in public databases through blastx. gene expression abundance under six conditions were quantified by calculating fpkm value. ultimately, we found genes at least 2-fold up-regulated while genes at least 2-fold down-regulated in nitrogen-limited condition. also, genes were at least 2-fold up-regulated in light while genes at least 2-fold down-regulated. finally, genes were selected to conduct rt-qpcr and genes showed the similar results as those identified by transcriptomic analysis in term of differential expression.  de novo transcriptomic analyses have generated enormous information over c. sorokiniana, revealing a broad overview of genomic information related to lipid accumulation and photosynthetic carbon fixation. the genes with expression change under different conditions are highly likely the potential targets for genetic modification to improve lipid production and co fixation efficiency in oleaginous microalgae. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chlorella sorokinianatranscriptomelipid accumulationphotosynthetic carbon fixationrna-seqnational basic research program of china 2011cb200900wang qinhong issue-copyright-statement© the author 2016 