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Cross-species dissection of saline-related genes by genetically deciphering a euryhaline microalga Chlorella sp.

ABSTRACT:

MEM25 and FACHB-9 respectively represent salt-tolerant and freshwater species of the genus Chlorella. By setting salinity of 35 and 105 as the high and low salinity conditions for MEM25, and salinity of 0 and 35 as the high and low salinity conditions for FACHB-9, we measured the metabolome data. To seek the difference in salt tolerance between two classic chlorella species. The measurement conditions are publicized as follows. Approximately 500 mg of algal biomass was collected, followed by metabolite extraction. Algal cells were suspended in 1 mL mixture of methanol and water (7/3, vol/vol) and kept at − 80 °C for 2 min. The internal standard used was 2-chloro-l-phenylalanine (0.3 mg mL-1). The mixture was vortexed at 60 Hz for 2 min, followed by ultrasonication at ambient temperature for 30 min. Samples were then centrifuged at 13,000 rpm, 4 °C for 15 min. The supernatants were collected using crystal syringes, filtered through 0.22 μm microfilters and transferred to LC vials, followed by storage at − 80 °C. LC–MS/MS analyses were performed using an UHPLC system (1290,Agilent Technologies).

INSTRUMENT(S): Liquid Chromatography MS - alternating - reverse-phase

PROVIDER: MTBLS13425 | MetaboLights | 2025-12-01

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS13425_LC-MS_alternating_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS13425_LC-MS_alternating_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular
	s_MTBLS13425.txt	Txt

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Project description:By applying Illumina Novaseq 6000, Chlorella sp. TLD6B cells of the control group on day zero and 18, as well as under low salt stress (NaCl1) and under high salt stress (NaCl2) on day 18 were selected for transcriptome sequencing analysis. Meanwhile, 0.05 g/mL ( PEG1) and 0.1 g/mL PEG-6000 (medium for drought stress, PEG2 ) were used to prepare the drought-stressed Chlorella sp. TLD6B cells. Each treatment had two replicates. Clean data were filtered after the removal of adapters, poly-N strands, and low-quality reads. There were no reference genomes for Chlorella sp. TLD6B, and de novo assembly for clean reads was performed by using Trinity. The sequences were compared with databases such as NR, NT, Swiss-Pro, GO, KEGG, PFAM, and KOG using Blast X (e-value ≤ 10-5). The GO annotation of unigenes was obtained using BLAST2GO. FPKM method was used for the analysis of gene expression levels (Trapnell et al., 2010). Out of six samples, a total of 963,078,184 raw reads were generated. A total of 947,225,244 clean reads were obtained based on the base quality score and read length. Meanwhile, the GC percentage in clean reads reached nearly 66.0%, with Q20 being above 96%. A total of 219,577 transcripts with an average length of 1,394 bp were obtained. In total, 155,503 non-redundant unigenes were assembled for the following analyses. The length of the unigenes ranged from 200 bp to 23,825 bp, with an average length of 1,842 bp. Under different salt stress, verification had been conducted with qRT-PCR on nine unigenes of different pathways, which were related to lipid metabolism. The detection results by qRT-PCR were highly correlated with RNA-Seq results (r = 0.890, r2 = 0.791), which indicated that the RNA-Seq data of Chlorella sp. TLD6B under salt stress were accurate and reliable. Our study represents the first detailed analysis of Chlorella sp. TLD6B under salt stress transcriptomes. Hierarchical clustering of differentially expressed genes uncovered several currently uncharacterized genes that may contribute to the function about lipid accumulation of Chlorella sp. TLD6B under salt stress.

Project description:Algae of the genus Dunaliella are known to be able to grow in a wide range of salinities. To investigate which genes may be important for growth under extremely low or high salinities, a survey of the Dunaliella sp. transcriptome was performed by microarray analysis. For the cDNA microarray 778 expressed sequence tags (ESTs) were generated from a cDNA library of Dunaliella sp. The cDNA microarray was used to compare the transcription profile of cells that were acclimated to extreme saline conditions (0.08 M and 4.5 M NaCl) with the transcription profile of cells acclimated to an intermediate salinity at 1.5 M NaCl. The comparative microarray analysis indicated that 211 out of the 778 ESTs differed in their expression levels more than two-fold in cells grown at extreme different salinity when compared to cells grown at intermediate salinity (1.5M NaCl). The sequences of these 211 cDNAs were classified into 169 non-redundant gene clusters. Of these genes, the expression of 36 genes was increased and 69 genes were suppressed in cells grown at low salinity. The expression of 49 genes was increased and 85 genes were suppressed in cells grown at high salinity. One-hundred thirty four genes have been revealed to show up- and down-regulation by high salinity (4.5M NaCl). Of the high salinity-regulated genes 52% were also influenced by low salinity (0.08M NaCl), revealing a dense overlap between low and high salt responses. Only 35 genes are regulated independently by low salt condition. Strong relationships, however, were observed in the expression of these extreme salinity-responsive genes based on Venn diagram analysis, which found 70 genes that responded to these two salinity conditions. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of the transcriptome in the microalga Dunaliella sp. and it provides a platform for further functional investigation of differentially expressed genes in Dunaliella. Keywords: Low salt(0.08M NaCl) or high salt(4.5M NaCl)-acclimated cells

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Cross-species dissection of saline-related genes by genetically deciphering a euryhaline microalga Chlorella sp.

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