Chaogang Wang

Genome-wide long non-coding RNA screening, identification and characterization in a model microorganism Chlamydomonas reinhardtii

Microalgae are regarded as the most promising biofuel candidates and extensive metabolic engineering were conducted but very few improvements were achieved. Long non-coding RNA (lncRNA) investigation and manipulation may provide new insights for this issue. LncRNAs refer to transcripts that are longer than 200 nucleotides, do not encode proteins but play important roles in eukaryotic gene regulation. However, no information of potential lncRNAs has been reported in eukaryotic alga. Recently, we performed RNA sequencing in Chlamydomonas reinhardtii, and obtained totally 3,574 putative lncRNAs. 1440 were considered as high-confidence lncRNAs, including 936 large intergenic, 310 intronic and 194 anti-sense lncRNAs. The average transcript length, ORF length and numbers of exons for lncRNAs are much less than for genes in this green alga. In contrast with human lncRNAs of which more than 98% are spliced, the percentage in C. reinhardtii is only 48.1%. In addition, we identified 367 lncRNAs responsive to sulfur deprivation, including 36 photosynthesis-related lncRNAs. This is the first time that lncRNAs were explored in the unicellular model organism C. reinhardtii. The lncRNA data could also provide new insights into C. reinhardtii hydrogen production under sulfur deprivation.

Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii

BackgroundNutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux.ResultsamiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16–28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7–48.6%, which mostly increased the content of C16–C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20–C22 fatty acid content significantly increased by 85–160%.ConclusionsOverall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids.

Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-head method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 mug/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.

Effect of overexpression of LPAAT and GPD1 on lipid synthesis and composition in green microalga Chlamydomonas reinhardtii

Biodiesel is an alternative energy source which has attracted increasing attention lately. Although algae-based biodiesel production has many benefits, it is still far from industrial application. Research suggests that improving lipid quality and production through genetic engineering of metabolic pathways will be the most promising way. To enhance lipid content, both lysophosphatidic acyltransferase gene (c-lpaat) and glycerol-3-phosphate dehydrogenase gene (c-gpd1), optimized according to the codon bias of Chlamydomonas reinhardtii, were inserted into the genomic DNA of model microalga C. reinhardtii by the glass bead method. Transgenic algae were screened by zeomycin resistance and RT-PCR. The transcription levels of inserted genes and the fatty acid content were significantly increased after intermittent heat shock. Most of all, the transcription levels of c-lpaat and c-gpd1 were increased 5.3 and 8.6 times after triple heat shocks, resulting in an increase of 44.5 and 67.5% lipid content, respectively. Furthermore, the content of long-chain saturated fatty acids and monounsaturated fatty acids, especially C18 and C18:1t, notably increased, while unsaturated fatty acids dramatically decreased. The results of this study offer a new strategy combining genetic manipulation and intermittent heat shock to enhance lipid production, especially the production of long-chain saturated fatty acids, using C. reinhardtii.

Identification and characterization of a novel defensin from Asian green mussel Perna viridis

ABSTRACT Defensin is one of the most diversified groups of antimicrobial peptides in invertebrate. In the present study, a novel defensin member referred as Pv‐Def was identified and characterized from Asian green mussel Perna viridis. Using in silico survey of several EST databases released from diverse tissues of P. viridis, a single peptide referred as Pv‐Def was predicted as defensin homologue with Mytilus counterparts. Further analysis on gene structure revealed that Pv‐Def was 1001nt in length and consisted of 3 exons and 2 introns. The precursor of Pv‐Def was composed of a signal peptide of 19 amino acids and a mature peptide of 45 amino acids. The mature Pv‐Def peptide contains 6 cysteines which formed 3 disulfide bonds at 27C1‐ 54C4, 40C2‐ 60C5 and 44C3‐ 62C6. Like most of the defensin family members, mature Pv‐Def peptide included an alpha helix and 2 beta strands. Pv‐Def showed significantly tissue‐specific expression pattern, while highest transcription level was observed in hepatopancreas, which was about 900 folds to that in hemocytes. Moreover, the expression of Pv‐Def mRNA in hemocytes was significantly and accurately up‐regulated at different time intervals by Vibrio parahaemolyticus challenge. Interestingly, phylogenetic analysis suggested that the Pv‐Def possesses closest relationships with arthropods counterparts rather than other mollusk defensins. To our knowledge, this is the first time that a defensin member was reported in Asian green mussel P. viridis. HIGHLIGHTSA novel defensin member (Pv‐Def) was identified from Perna viridis.Pv‐Def is 1001nt in length and consisted of 3 exons and 2 introns.The precursor of Pv‐Def was composed of a signal peptide of 19 amino acids and a mature peptide of 45 amino acids.Pv‐Def mRNA was highly expressed in hepatopancreas and could be induced accurately by Vibrio parahaemolyticus challenge.Pv‐Def possesses closest relationships with arthropods counterparts rather than other mollusk defensins.

Improved photobio-H 2 production regulated by artificial miRNA targeting psbA in green microalga Chlamydomonas reinhardtii

BackgroundSulfur-deprived cultivation of Chlamydomonas reinhardtii, referred as “two-stage culture” transferring the cells from regular algal medium to sulfur-deplete one, has been extensively studied to improve photobio-H2 production in this green microalga. During sulfur-deprivation treatment, the synthesis of a key component of photosystem II complex, D1 protein, was inhibited and improved photobio-H2 production could be established in C. reinhardtii. However, separation of algal cells from a regular liquid culture medium to a sulfur-deprived one is not only a discontinuous process, but also a cost- and time-consuming operation. More applicable and economic alternatives for sustained H2 production by C. reinhardtii are still highly required.ResultsIn the present study, a significant improvement in photobio-H2 production was observed in the transgenic green microalga C. reinhardtii, which employed a newly designed strategy based on a heat-inducible artificial miRNA (amiRNA) expression system targeting D1-encoded gene, psbA. A transgenic algal strain referred as “amiRNA-D1” has been successfully obtained by transforming the expression vector containing a heat-inducible promoter. After heat shock conducted in the same algal cultures, the expression of amiRNA-D1 was detected increased 15-fold accompanied with a 73% decrease of target gene psbA. More interestingly, this transgenic alga accumulated about 60% more H2 content than the wild-type strain CC-849 at the end of 7-day cultivation.ConclusionsThe photobio-H2 production in the engineered transgenic alga was significantly improved. Without imposing any nutrient-deprived stress, this novel strategy provided a convenient and efficient way for regulation of photobio-H2 production in green microalga by simply “turn on” the expression of a designed amiRNA.

Expression and molecular analysis ofphbB gene inChlamydomonas reinhardtii

The expression vector containingphbB andble genes was constructed and transformed into cell-walldeficient strainChlamydomonas reinhardtii CC-849 by the glass-bead method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 μg/mL Zeomycin. Transgenic alga, which could expressphbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.

Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii

WD40-repeat (WDR) domain-containing proteins are subunits of multi-protein E3 ligase complexes regulating various cellular and developmental activities in eukaryotes. Chlamydomonas reinhardtii serves as a model organism to study lipid metabolism in microalgae. Under nutrition deficient conditions, C. reinhardtii accumulates lipids for survival. The proteins in C. reinhardtii flagella have diverse functions, such as controlling the motility and cell cycle, and environment sensing. Here, we characterized the function of CrFAP89, a flagella-associated WDR-containing protein, which was identified from C. reinhardtii nitrogen deficiency transcriptome analysis. Quantitative real time-PCR showed that the transcription levels of CrFAP89 were significantly enhanced upon nutrient deprivation, including nitrogen, sulfur, or iron starvation, which is considered an effective condition to promote triacylglycerol (TAG) accumulation in microalgae. Under sulfur starvation, the expression of CrFAP89 was 32.2-fold higher than the control. Furthermore, two lines of RNAi mutants of CrFAP89 were generated by transformation, with gene silencing of 24.9 and 16.4%, respectively. Inhibiting the expression of the CrFAP89 gene drastically increased cell density by 112–125% and resulted in larger cells, that more tolerant to nutrition starvation. However, the content of neutral lipids declined by 12.8–19.6%. The fatty acid content in the transgenic algae decreased by 12.4 and 13.3%, mostly decreasing the content of C16:0, C16:4, C18, and C20:1 fatty acids, while the C16:1 fatty acid in the CrFAP89 RNAi lines increased by 238.5 to 318.5%. Suppressed expression of TAG biosynthesis-related genes, such as CrDGAT1 and CrDGTTs, were detected in CrFAP89 gene silencing cells, with a reduction of 16–78%. Overall our results suggest that down-regulating of the expression of CrFAP89 in C. reinhardtii, resulting in an increase of cell growth and a decrease of fatty acid synthesis with the most significant decrease occurring in C16:0, C16:4, C18, and C20:1 fatty acid. CrFAP89 might be a regulator for lipid accumulation in C. reinhardtii.