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De novo transcriptomic analysis of Chlorella sorokiniana reveals differential genes expression in photosynthetic carbon fixation and lipid production

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Results: Six cultivation conditions were selected to cultivate C. sorokiniana. Lipid content of C. sorokiniana under nitrogen-limited condition was 2.96 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 244,291,069 raw reads with length of 100 bp were produced. After preprocessed, these reads were de novo assembled into 63,811 contigs among which 23,528 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 385 genes at least 2-fold up-regulated while 71 genes at least 2-fold down-regulated in nitrogen-limited condition. Also, 204 genes were at least 2-fold up-regulated in light while 638 genes at least 2-fold down-regulated. Finally, 16 genes were selected to conduct RT-qPCR and 15 genes showed the similar results as those identified by transcriptomic analysis in term of differential expression.

Conclusions: 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 CO2 fixation efficiency in oleaginous microalgae.

Electronic supplementary material: The online version of this article (doi:10.1186/s12866-016-0839-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Venn diagram of annotated contigs’ number in different database. Nr: NCBI’s non-redundant database. COG: Clusters of Orthologous Groups (COG) database. EC number and KO: the annotation results of KEGG database. Each figure means the number of annotated contigs in corresponding databases
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Fig3: Venn diagram of annotated contigs’ number in different database. Nr: NCBI’s non-redundant database. COG: Clusters of Orthologous Groups (COG) database. EC number and KO: the annotation results of KEGG database. Each figure means the number of annotated contigs in corresponding databases

Mentions: After compared against the NCBI’s Nr database using Blastx, 23,496 contigs (36.8 % of total contigs) were found having homologous sequence in Nr database (Fig. 3, Additional file 4). Due to the lack of genome information, a large proportion of the contigs (40298, 63.2 %) could not be matched to homologous sequence in any database, among which 10,471 potential coding regions were predicted using Transdecoder (Additional file 5). 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 2,789 contigs were assigned with EC number (Fig. 3, Additional file 4). There were 2,371 contigs which were all matched with homologous sequences in all the databases used (Fig. 3). Particularly, the length of most contigs with homologous sequence in Nr database were between 500 and 2500 bp (14801, 62.79 %) and the match efficiency decreased as the length of contigs increased (Fig. 2b), 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 (80.24 % of all annotated contigs), Coccomyxa subellipsoidea C-169 (6.86 %) and Volvox carteri f. nagariensis (2.03 %) (Fig. 2c), 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 [16] and Chlamydomonas moewusii [20]. The E-value distribution of the top match in Nr database showed that 55.13 % of the matched sequences had E-value ≤ 1.0E-50, and 44.87 % ranged from 1.0E-5 to 1.0E-50 (Fig. 2d). Similar results were also reported in the de novo transcriptomic analysis of Ambystoma mexicanum [21].Fig. 3


De novo transcriptomic analysis of Chlorella sorokiniana reveals differential genes expression in photosynthetic carbon fixation and lipid production
Venn diagram of annotated contigs’ number in different database. Nr: NCBI’s non-redundant database. COG: Clusters of Orthologous Groups (COG) database. EC number and KO: the annotation results of KEGG database. Each figure means the number of annotated contigs in corresponding databases
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037625&req=5

Fig3: Venn diagram of annotated contigs’ number in different database. Nr: NCBI’s non-redundant database. COG: Clusters of Orthologous Groups (COG) database. EC number and KO: the annotation results of KEGG database. Each figure means the number of annotated contigs in corresponding databases
Mentions: After compared against the NCBI’s Nr database using Blastx, 23,496 contigs (36.8 % of total contigs) were found having homologous sequence in Nr database (Fig. 3, Additional file 4). Due to the lack of genome information, a large proportion of the contigs (40298, 63.2 %) could not be matched to homologous sequence in any database, among which 10,471 potential coding regions were predicted using Transdecoder (Additional file 5). 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 2,789 contigs were assigned with EC number (Fig. 3, Additional file 4). There were 2,371 contigs which were all matched with homologous sequences in all the databases used (Fig. 3). Particularly, the length of most contigs with homologous sequence in Nr database were between 500 and 2500 bp (14801, 62.79 %) and the match efficiency decreased as the length of contigs increased (Fig. 2b), 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 (80.24 % of all annotated contigs), Coccomyxa subellipsoidea C-169 (6.86 %) and Volvox carteri f. nagariensis (2.03 %) (Fig. 2c), 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 [16] and Chlamydomonas moewusii [20]. The E-value distribution of the top match in Nr database showed that 55.13 % of the matched sequences had E-value ≤ 1.0E-50, and 44.87 % ranged from 1.0E-5 to 1.0E-50 (Fig. 2d). Similar results were also reported in the de novo transcriptomic analysis of Ambystoma mexicanum [21].Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Results: Six cultivation conditions were selected to cultivate C. sorokiniana. Lipid content of C. sorokiniana under nitrogen-limited condition was 2.96 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 244,291,069 raw reads with length of 100 bp were produced. After preprocessed, these reads were de novo assembled into 63,811 contigs among which 23,528 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 385 genes at least 2-fold up-regulated while 71 genes at least 2-fold down-regulated in nitrogen-limited condition. Also, 204 genes were at least 2-fold up-regulated in light while 638 genes at least 2-fold down-regulated. Finally, 16 genes were selected to conduct RT-qPCR and 15 genes showed the similar results as those identified by transcriptomic analysis in term of differential expression.

Conclusions: 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 CO2 fixation efficiency in oleaginous microalgae.

Electronic supplementary material: The online version of this article (doi:10.1186/s12866-016-0839-8) contains supplementary material, which is available to authorized users.

No MeSH data available.