Zhenghong Sui

Inhibition of pds gene expression via the RNA interference approach in Dunaliella salina (Chlorophyta).

To investigate the potential of double-stranded RNA interferencing with gene expression in Dunaliella salina, a plasmid pBIRNAI-Dsa was constructed to express hairpin RNA (hpRNA) containing sequences homologous to phytoene desaturase gene (pds), a key gene in carotenoid biosynthesis, and transformed into D. salina by electroporation. The relative transcription level of pds in pBIRNAI-Dsa–treated cells to nontreated cells was quantitated and the gene silencing efficiency by RNAi was evaluated via real-time polymerase chain reaction (PCR). The transcriptions of pds of the pBIRNAI-Dsa–treated group changed compared to those of the control group, and the

Genome survey sequencing and genetic background characterization of Gracilariopsis lemaneiformis (Rhodophyta) based on next-generation sequencing.

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Comparison of potential diatom ‘barcode’ genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta

was lowest on the 7th day, corresponding to 0.281265-fold of the relative pds control transcript; a relatively strong gene inhibition effect was therefore deduced. The transcript of pds may be modulated in a wide range, and a reduced transcription even to 28% of the normal level may be tolerated for its survival. This study shows that dsRNA-mediated genetic interference can induce sequence-specific inhibition of gene expression and pBIRNAI-Dsa can be used for transient suppression of gene expression in D. salina. The aim of this study was to exploit dsRNA-mediated gene silencing and to provide a foundation for gene function research in D. salina.

Cloning and Characterization of hoxH Genes from Arthrospira and Spirulina and Application in Phylogenetic Study

In this article, high-throughput de novo transcriptomic sequencing was performed in Alexandrium catenella, which provided the first view of the gene repertoire in this dinoflagellate based on next-generation sequencing (NGS) technologies. A total of 118,304 unigenes were identified with an average length of 673bp (base pair). Of these unigenes, 77,936 (65.9%) were annotated with known proteins based on sequence similarities, among which 24,149 and 22,956 unigenes were assigned to gene ontology categories (GO) and clusters of orthologous groups (COGs), respectively. Furthermore, 16,467 unigenes were mapped onto 322 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). We also detected 1143 simple sequence repeats (SSRs), in which the tri-nucleotide repeat motif (69.3%) was the most abundant. The genetic facts and significance derived from the transcriptome dataset were suggested and discussed. All four core nucleosomal histones and linker histones were detected, in addition to the unigenes involved in histone modifications.190 unigenes were identified as being involved in the endocytosis pathway, and clathrin-dependent endocytosis was suggested to play a role in the heterotrophy of A. catenella. A conserved 22-nt spliced leader (SL) was identified in 21 unigenes which suggested the existence of trans-splicing processing of mRNA in A. catenella.

Cloning and analysis of the galactose-1-phosphate uridylyltransferase (galt) gene of Gracilariopsis lemaneiformis (Rhodophyta) and correlation between gene expression and agar synthesis

Gracilariopsis lemaneiformis has a high economic value and is one of the most important aquaculture species in China. Despite it is economic importance, it has remained largely unstudied at the genomic level. In this study, we conducted a genome survey of Gp. lemaneiformis using next-generation sequencing (NGS) technologies. In total, 18.70 Gb of high-quality sequence data with an estimated genome size of 97 Mb were obtained by HiSeq 2000 sequencing for Gp. lemaneiformis. These reads were assembled into 160,390 contigs with a N50 length of 3.64 kb, which were further assembled into 125,685 scaffolds with a total length of 81.17 Mb. Genome analysis predicted 3490 genes and a GC% content of 48%. The identified genes have an average transcript length of 1,429 bp, an average coding sequence size of 1,369 bp, 1.36 exons per gene, exon length of 1,008 bp, and intron length of 191 bp. From the initial assembled scaffold, transposable elements constituted 54.64% (44.35 Mb) of the genome, and 7737 simple sequence repeats (SSRs) were identified. Among these SSRs, the trinucleotide repeat type was the most abundant (up to 73.20% of total SSRs), followed by the di- (17.41%), tetra- (5.49%), hexa- (2.90%), and penta- (1.00%) nucleotide repeat type. These characteristics suggest that Gp. lemaneiformis is a model organism for genetic study. This is the first report of genome-wide characterization within this taxon.

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The agarophyte Gracilariopsis lemaneiformis is both important for biological research and of significant economic value. However, the genetic diversity of wild populations of the alga has not been studied. We used amplified fragment length polymorphism (AFLP) PCR and simple sequence repeat (SSR) analysis to investigate diversity in four field populations, three from the coast of Qingdao and one from Weihai, China. Forty G. lemaneiformis isolates collected from the four different geographical groups were analyzed using 16 pairs of SSR primers for PCR amplification. However, no polymorphisms were detected, indicative of a degree of genetic homogeneity. A total of 347 reproducible bands were then amplified using eight AFLP primer pairs, and genetic indices of diversity within and between populations were calculated. This analysis revealed only low levels of genetic diversity both within and between the four geographical groups of G. lemaneiformis. The Weihai population showed a higher within-population genetic diversity than any of the Qingdao populations. The data suggest that there is only limited gene flow between populations. An UPGMA dendrogram revealed two main clusters, and one of these included all the Qingdao isolates. The order of clustering was in accordance with their geographical distribution. These results suggest that the wild G. lemaneiformis populations are closely related and that there is little genetic diversity within wild germplasm in the regions sampled.

Identification of phase relative genes in tetrasporophytes and female gametophytes of Gracilaria/Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta)

Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

Isolation and characterization of calmodulin gene of Alexandrium catenella (Dinoflagellate) and its performance in cell growth and heat stress

Partial hoxH genes of 2 cyanobacterial genera, including 5 strains of Arthrospira and 2 strains of Spirulina, were cloned and characterized. This gene encodes the large subunit of nickel-iron hydrogenase. The results showed that these genes comprised 1349 nucleotides in Arthrospira and 1343 nucleotides in Spirulina, respectively. The GC contents of hoxH were 45.7% to 47.3% in Arthrospira and up to 50.4% to 50.9% in Spirulina. The hoxH gene was demonstrated to be single copy by Southern analysis, and its transcription was verified by reverse transcriptase polymerase chain reaction in Arthrospira platensis FACHB341. The similarities of nucleotide sequences among the 5 strains of Arthrospira ranged from 95.7% to 99.8%, which are higher than those between Arthrospira and Spirulina (72.9–77.0%). However, similarity between the 2 Spirulina strains was only 72.5%, slightly lower than that between the 2 genera. A phylogenetic tree based on hoxH was constructed. All 5 strains of Arthrospira formed a monophyletic clade, which was highly supported by bootstrap value, while the 2 strains of Spirulina were separated into 2 different clades.