Exploring the key genes of fucoxanthin biosynthesis in Phaeodactylum tricornutum under phosphorus deficiency, red light and yellow light using WGCNA.

Weighted gene co-expression network analysis (WGCNA) is a method for analysing gene expression patterns across multiple samples, clustering genes with similar expression patterns and identifying key genes associated with specific traits or phenotypes. In this study, we investigated the effects of fucoxanthin accumulation in Phaeodactylum tricornutum in response to abiotic stresses of phosphorus deficiency, red light, and yellow light using transcriptome sequencing and weighted gene co-expression network analysis. The results showed that compared to the control, the fucoxanthin content of P. tricornutum was significantly increased after phosphorus deficiency and red light treatment (P<0.05), but significantly decreased after yellow light treatment (P<0.05). A weighted gene co-expression network was constructed using 10,392 genes obtained from transcriptome sequencing, and ß=18 (R2>0.8) was chosen as a soft threshold in order to ensure a scale-free network. A total of 10 co-expression modules were identified by correlation analysis of fucoxanthin content, with the purple module positively correlated with fucoxanthin content (r=0.9, P=1E-200), and 9 key genes were identified, including five genes in the fucoxanthin biosynthesis pathway (DXR, PSY, PDS1, ZEP2, VDL2) and 4 transcription factors (bHLH5, HOX2, CCHH13, HSF1b). Further qRT-PCR confirmed that key genes were more highly expressed in the phosphorus deficiency treatment and linear regression analysis showed that the relative gene expressions were all highly correlated with the transcriptome data. The results of this study provide a basis for further investigation of the complex regulatory mechanisms of fucoxanthin in P. tricornutum.

Expression analysis of different ß-carotenoid hydroxylase gene families in stress response in Dunaliella viridis.

ß-carotenoid hydroxylase (CHYB) is an important rate-limiting enzyme in the biosynthesis of plant carotenoid. In this study, chyb1 and chyb2, two gene families in Dunaliella viridis were obtained by RNA-seq. The fragment of promoters of CHYB family genes, 1 080 bp for chyb1 (GenBank No. KY012338) and 1 155 bp for chyb2 (GenBank No. KY012339) were cloned by the Genome Walking Technology, respectively. Cis-acting elements of two promoters were analyzed by Plantcare soft. The results show that the chyb1 gene promoter contains more cis-acting elements in responses to abiotic stresses, such as methyl jasmonate, arachidonic acid, acetylsalicylic acid, and so on. On the other hand, the chyb2 promoter contains more cis-acting elements in response to light stress. qRT-PCR results show that the mRNA expression levels of CHYBs are modulated by their promoters, and different CHYB gene families response to distinct stresses.

[Transcriptome analysis of Dunaliella viridis].

In order to understand the gene information, function, haloduric pathway (glycerolipid metabolism) and related key genes for Dunaliella viridis, we used Illumina HiSeqTM 2000 high-throughput sequencing technology to sequence its transcriptome. Trinity soft was used to assemble the data to form transcripts. Based on the Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG ) databases, we carried out functional annotation and classification, pathway annotation, and the opening reading fragment (ORF) sequence prediction of transcripts. The key genes in the glycerolipid metabolism were analyzed. The results suggested that 81,593 transcripts were found, and 77,117 ORF sequences were predicted, accounting for 94.50% of all transcripts. COG classification results showed that 16,569 transcripts were assigned to 24 categories. GO classification annotated 76,436 transcripts. The number of transcripts for biologcial processes was 30,678, accounting for 40.14% of all transcripts. KEGG pathway analysis showed that 26,428 transcripts were annotated to 317 pathways, and 131 pathways were related to metabolism, accounting for 41.32% of all annotated pathways. Only one transcript was annotated as coding the key enzyme dihydroxyacetone kinase involved in the glycerolipid pathway. This enzyme could be related to glycerol biosynthesis under salt stress. This study further improved the gene information and laid the foundation of metabolic pathway research for Dunaliella viridis.

[Cloning and expression analysis of the NFκB inhibitor IκBα of ayu (Plecoglossus altivelis)].

The NFκB inhibitor (IκBα) is an integral part of NFκB/IκB signaling pathways, which plays roles in a variety of immune responses, such as bacterial infection resistance. By interacting with nuclear transcription factor NFκB, IκBα controls a variety of biological immune gene expressions. In this study, full-length cDNA (1341 bp) of the NFκB inhibitor IκBα (PaIκBα, GenBank Accession No. JN801027) of Plecoglossus altivelis was obtained by RACE and PCR, and included a 5' untranslated region (UTR) (64 bp), a 3' untranslated region (UTR) (341 bp) and an open reading frame (ORF) (936 bp) encoding a polypeptide of 311 amino acids. PaIκBα had high homology with other IκBαs, containing a conserved ankyrin repeat domain, which was required for interacting with NFκB, a PEST sequence in the C-terminus and a signal responsive domain in the N-terminus. The deduced amino acid sequence of PaIκBα shared 95% homology with Osmerus mordax, and 76%, 75%, 70%, and 68% homology with Salmo salar, Oncorhynchus mykiss, Nile tilapia, and Siniperca chuatsi, respectively. Phylogenetic analysis revealed that IκBα of ayu and Osmerus mordax, Salmo salar, Oncorhynchus mykiss, Nile tilapia, and Siniperca chuatsi were in the same phylogenetic tree. RT-PCR analysis showed that PaIκBα mRNA expression was highest in the liver, kidney, intestine, and gills, then followed by the spleen, brain and muscle, and was lowly expressed in the heart. Likewise, after Aeromonas hydrophila infection, the mRNA level of ayu PaIκBα in the liver was also up-regulated.

Molecular cloning and expression profile analysis of Ginkgo biloba DXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway.

Plant diterpenes such as ginkgolides are biosynthesized via the recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The initial step of the MEP pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), which may thus be considered the first committed step of the MEP pathway for ginkgolides biosynthesis. The full-length cDNA of DXS was isolated and characterized from the gymnosperm plant species, Ginkgo biloba. The full-length cDNA of GbDXS was 2795 bp containing a 2154 bp open reading frame (ORF) encoding 717 amino acids. Comparative and bioinformatic analyses revealed that GbDXS has extensive homology with DXSs from other plant species and, like these, contains a conserved transit peptide for plastid import, histidine residue, a putative thiamine diphosphate-binding site and a transketolase motif. Phylogenetic analysis indicates that GbDXS belongs to the plant DXS1 cluster and suggests it to be more ancient than other plant DXSs. GbDXS was found to be expressed in all tested tissues including roots, stems, leaves, pericarps and seeds. Expression profiling analyses revealed that GbDXS expression was induced by exogenous elicitors including methyl jasmonate, arachidonic acid, acetylsalicylic acid and ceric ammonium sulfate, and showed that the transcription levels were correlated with ginkgolide accumulation, suggesting that DXS might play a regulatory role in ginkgolide biosynthesis in cell culture of G. biloba at the transcriptional level.