Mutagenesis and fluorescence-activated cell sorting of oleaginous Saccharomyces cerevisiae and the multi-omics analysis of its high lipid accumulation mechanisms.

Acquiring lipid-producing strains of Saccharomyces cerevisiae is necessary for producing high-value palmitoleic acid. This study sought to generate oleaginous S. cerevisiae mutants through a combination of zeocin mutagenesis and fluorescence-activated cell sorting, and then to identify key mutations responsible for enhanced lipid accumulation by multi-omics sequencing. Following three consecutive rounds of mutagenesis and sorting, a mutant, MU310, with the lipid content of 44%, was successfully obtained. Transcriptome and targeted metabolome analyses revealed that a coordinated response involving fatty acid precursor biosynthesis, nitrogen metabolism, pentose phosphate pathway, ethanol conversion, amino acid metabolism and fatty acid ß-oxidation was crucial for promoting lipid accumulation. The carbon fluxes of acetyl-CoA and NADPH in lipid biosynthesis were boosted in these pathways. Certain transcriptional regulators may also play significant roles in modulating lipid biosynthesis. Results of this study provide high-quality resource for palmitoleic acid production and deepen the understanding of lipid synthesis in yeast.

Full-length transcriptome analysis of a bloom-forming dinoflagellate Prorocentrum shikokuense (Dinophyceae).

Prorocentrum shikokuense (formerly P. donghaiense) is a pivotal dinoflagellate species associating with the HABs in the East China Sea. The complexity of its large nuclear genome hindered us from understanding its genomic characteristics. Full-length transcriptome sequencing offers a practical solution to decipher the physiological mechanisms of a species without the reference genome. In this study, we employed single-molecule real-time (SMRT) sequencing technology to sequence the full-length transcriptome of Prorocentrum shikokuense. We successfully generated 41.73 Gb of clean SMRT sequencing reads and isolated 105,249 non-redundant full-length non-chimeric reads. Our trial has led to the identification of 11,917 long non-coding RNA transcripts, 514 alternative splicing events, 437 putative transcription factor genes from 17 TF gene families, and 34,723 simple sequence repeats. Additionally, a total of 78,265 open reading frames were identified, of them 15,501 were the protein coding sequences. This dataset is valuable for annotating P. shikokuense genome, and will contribute significantly to the in-depth studies on the molecular mechanisms underlining the dinoflagellate bloom formation.

Pioneering DNA assembling techniques and their applications in eukaryotic microalgae.

Assembling DNA fragments is a fundamental manipulation of cloning microalgal genes and carrying out microalgal synthetic biological studies. From the earliest DNA recombination to current trait and metabolic pathway engineering, we are always accompanied by homology-based DNA assembling. The improvement and modification of pioneering DNA assembling techniques and the combinational applications of the available assembling techniques have diversified and complicated the literature environment and aggravated our identification of the core and pioneering methodologies. Identifying the core assembling methodologies and using them appropriately and flourishing them even are important for researchers. A group of microalgae have been evolving as the models for both industrial applications and biological studies. DNA assembling requires researchers to know the methods available and their improvements and evolvements. In this review, we summarized the pioneering (core; leading) DNA assembling techniques developed previously, extended these techniques to their modifications, improvements and their combinations, and highlighted their applications in eukaryotic microalgae. We predicted that the gene(s) will be assembled into a functional cluster (e.g., those involving in a metabolic pathway, and stacked on normal microalgal chromosomes, their artificial episomes and looming artificial chromosomes. It should be particularly pointed out that the techniques mentioned in this review are classified according to the strategy used to assemble the final construct.

Macropinocytosis in Gracilariopsis lemaneiformis (Rhodophyta).

Macropinocytosis is an endocytic process that plays an important role in animal development and disease occurrence but until now has been rarely reported in organisms with cell walls. We investigated the properties of endocytosis in a red alga, Gracilariopsis lemaneiformis. The cells non-selectively internalized extracellular fluid into large-scale endocytic vesicles (1.94 ± 0.51 µm), and this process could be inhibited by 5-(N-ethyl-N-isopropyl) amiloride, an macropinocytosis inhibitor. Moreover, endocytosis was driven by F-actin, which promotes formation of ruffles and cups from the cell surface and facilitates formation of endocytotic vesicles. After vesicle formation, endocytic vesicles could be acidified and acquire digestive function. These results indicated macropinocytosis in G. lemaneiformis. Abundant phosphatidylinositol kinase and small GTPase encoding genes were found in the genome of this alga, while PI3K, Ras, and Rab5, the important participators of traditional macropinocytosis, seem to be lacked. Such findings provide a new insight into endocytosis in organisms with cell walls and facilitate further research into the core regulatory mechanisms and evolution of macropinocytosis.

UDP-glucose pyrophosphorylase as a target for regulating carbon flux distribution and antioxidant capacity in Phaeodactylum tricornutum.

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme for polysaccharide synthesis, and its role in plants and bacteria is well established; however, its functions in unicellular microalgae remain ill-defined. Here, we perform bioinformatics, subcellular localization as well as in vitro and in vivo analyses to elucidate the functions of two UGPs (UGP1 and UGP2) in the model microalga Phaeodactylum tricornutum. Despite differences in amino acid sequence, substrate specificity, and subcellular localization between UGP1 and UGP2, both enzymes can efficiently increase the production of chrysolaminarin (Chrl) or lipids by regulating carbon flux distribution without impairing growth and photosynthesis in transgenic strains. Productivity evaluation indicate that UGP1 play a bigger role in regulating Chrl and lipid production than UGP2. In addition, UGP1 enhance antioxidant capacity, whereas UGP2 is involved in sulfoquinovosyldiacylglycerol (SQDG) synthesis in P. tricornutum. Taken together, the present results suggest that ideal microalgal strains can be developed for the industrial production of Chrl or lipids and lay the foundation for the development of methods to improve oxidative stress tolerance in diatoms.

Producing high value unsaturated fatty acid by whole-cell catalysis using microalga: A case study with Tribonema minus.

High value unsaturated fatty acids can be produced by de novo synthesis in microalgal cells, especially via heterotrophic cultivation. Unfortunately, the lipid accumulation of heterotrophic microalgae cannot be improved efficiently in conventional ways. Here we reported heterotrophic Tribonema minus, a promising resource for the production of palmitoleic acid which has increasing demands in health service for patients with metabolic syndrome, as whole-cell biocatalyst to develop a novel way of shifting low value exogenous saturated fatty acids to high value ones. Results showed that myristic acid is the best precursor for whole-cell catalysis; it elevated the lipid content of T. minus to 42.2%, the highest among the tried precursors. The influences of cultivation condition on the utilization of extrinsic myristic acid and lipid accumulation were also determined. Under the optimized condition, the lipid content reached as high as 48.9%. In addition, our findings showed that ~13.0% of C16:1 in T. minus is derived from extrinsic myristic acid, and 30.1% of metabolized precursor is converted into heterologous fatty acids. Thus, a feasible approach for both increasing the value of low value saturated fatty acid by bioconversion and enhancing the lipid accumulation in microalgae is proposed by supplementing extrinsic myristic acid.

How do microalgae in response to biological pollution treat in cultivation? A case study investigating microalgal defense against ciliate predator Euplotes vannus.

Microalgae have significant amounts of proteins, lipids, carotenoids, vitamins, minerals, and unique pigments. However, with the gradual expansion of microalgae cultivation, hostile biological pollution seriously restricted the large-scale microalgae cultivation and limited the exploitation of its biological resources. Moreover, protozoan poses the greatest threat to microalgae cultivation. Here, the relationship between six marine economic microalgae populations and their ciliate predator Euplotes vannus was examined. And four concentrations were designed for each type of microalgae to carry out the experiment. It was revealed that four species of microalgae inhibit the ciliate population growth at high density. Furthermore, the experiment which was the influence of microalgae at three different growth stages on the growth of the ciliates for these four kinds of high-density inhibitory microalgae was designed. The microalgae inhibitory effects were already exhibited at the end of the exponential growth phase, and it was significantly inhibited during the stationary growth phase. As the microalgae concentration increased, the inhibitory effect became more pronounced. This study provides fundamental data for screening protozoan-inhibiting microalgae and shows potential to be used in algae cultivation.

A review on the progress, challenges and prospects in commercializing microalgal fucoxanthin.

Fucoxanthin, the most abundant but nearly untapped carotenoid resource, is in the spotlight in the last decade from various perspectives due to a wide range of bioactivities and healthy benefits. The exploitation of fucoxanthin for nutraceutical and pharmaceutical purposes encompasses enormous scientific and economic potentials. Traditional production of fucoxanthin from brown algae (macroalgae) is constrained by limited yield and prohibitively high cost. Microalgae, as the most diverse photoautotrophs, hold the promises as sustainable sources and ideal cell factories for commercial fucoxanthin production, owing to their rich fucoxanthin content and excellent biomass productivity. In this work, the recent progress in upstream (microalgae selection, optimization of culture conditions, trophic modes, cultivation strategies and biosynthesis pathway) as well as downstream processes (extraction) of fucoxanthin production has been comprehensively and critically reviewed. The major bottlenecks, such as screening of fucoxanthin-producers, conflict between biomass and fucoxanthin accumulation under high light condition, unclear steps in biosynthesis pathway and limited evaluation of outdoor scale-up cultivation and extraction, have been pinpointed. Most importantly, the applications of emerging and conventional techniques facilitating commercialization of microalgal fucoxanthin are highlighted. The reviewed and evaluated include breeding and high-throughput screening methods of elite strains; flashing light effect inducing concurrent biomass and fucoxanthin accumulation; fucoxanthin biosynthesis and the regulatory mechanisms associating with its accumulation elucidated with the development of genetic engineering and omics techniques; and photobioreactors, harvesting and extraction techniques suitable for scaling up fucoxanthin production. In conclusion, the prospects of microalgal fucoxanthin commercialization can be expected with the joint development of fundamental phycology and biotechnology.

Potential to resist biological contamination in marine microalgae culture: Effect of extracellular substances of Nannochloropsis oceanica on population growth of Euplotes vannus and other protozoa.

The commercially important marine microalgae Nannochloropsis oceanica is easily ingested by protozoan predators during large-scale cultivation. However, investigations into the effect of microalgae on the growth of protozoa are scant. A feeding experiment was conducted with Euplotes vannus grazing on different concentrations of N. oceanica. The ciliate population was significantly lower in the high concentration of algae than that in the low or medium algal concentration treatments. The density of ciliates cultured in algae filtrate media was significantly lower than that in lysate media and the blank control. Furthermore, the algal cell filtrate was added to three other protozoan populations, and they all gradually lost their ability to move and their body shape changed. This study investigated the interactions between N. oceanica and protozoan predators and provides insight on using microalgal extracellular substances to control biological contamination in the future.

Uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome activation.

BACKGROUND AND AIM: Intestinal epithelial dysfunction is the foundation of various intestinal and extra-intestinal diseases, while the effects and mechanism of uric acid on the intestinal barrier are little known. TSPO has been shown to be related to the generation of ROS and is involved in regulating inflammation, whether uric acid drives intestinal epithelial dysfunction through TSPO-mediated NLRP3 inflammasome activation is unknown. METHODS: UOX gene knockout mouse (UOX-/-) were used for models of hyperuricemia. Fluorescein isothiocyanate (FITC)-labeled dextran was used to assess in vivo intestinal permeability. Serum lipopolysaccharide (LPS) and culture supernatants IL-1ß were measured using ELISA Kit. IEC-6 exposed to different concentrations of uric acid was used for in vitro experiment. Protein content and mRNA were assessed using Western blotting and Q-PCR, respectively. Intracellular ROS was determined using flow cytometry and fluorescence microscope. Mitochondrial membrane potential was detected on an immunofluorescence. Small interfering RNA transfection was used to assess the interaction between translocator protein (TSPO) and NLRP3 inflammasome. N-acetyl-L-cysteine (NAC) was used as ROS scavenger. RESULTS: Our results showed that hyperuricemia mice were characteristic by increased intestinal permeability. Hyperuricemia upregulated TSPO, increased production of ROS and activated NLRP3 inflammasome, which resulted in lower expression of occludin and claudin-1. In vitro, we showed that soluble uric acid alone increased the expression of TSPO, depolarized mitochondrial membrane potential, increased ROS release and activated NLRP3 inflammasome, which further reduced the expression of occludin and claudin-1. Silencing TSPO suppressed NLRP3 inflammasome activation and increased expression of claudin-1 and occludin, which was accompanied by lower levels of ROS. Scavenging ROS also significantly inhibited NLRP3 inflammasome activation without change of TSPO, indicating that TSPO-mediated NLRP3 inflammasome activation was dependent on ROS. CONCLUSIONS: In conclusion, uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome.

Association of Hyperuricemia With Immune Disorders and Intestinal Barrier Dysfunction.

BACKGROUND: More than 30-40% of uric acid is excreted via the intestine, and the dysfunction of intestinal epithelium disrupts uric acid excretion. The involvement of gut microbiota in hyperuricemia has been reported in previous studies, but the changes and mechanisms of intestinal immunity in hyperuricemia are still unknown. METHODS: This study developed a urate oxidase (Uox)-knockout (Uox-/-) mouse model for hyperuricemia using CRISPR/Cas9 technology. The lipometabolism was assessed by measuring changes in biochemical indicators. Furthermore, 4-kDa fluorescein isothiocyanate-labeled dextran was used to assess gut barrier function. Also, 16S rRNA sequencing was performed to examine the changes in gut microbiota in mouse feces. RNA sequencing, Western blot, Q-PCR, ELISA, and immunohistochemical analysis were used for measuring gene transcription, the number of immune cells, and the levels of cytokines in intestinal tissues, serum, kidney, liver, pancreas, and vascellum. RESULTS: This study showed that the abundance of inflammation-related microbiota increased in hyperuricemic mice. The microbial pattern recognition-associated Toll-like receptor pathway and inflammation-associated TNF and NF-kappa B signaling pathways were significantly enriched. The increased abundance of inflammation-related microbiota resulted in immune disorders and intestinal barrier dysfunction by upregulating TLR2/4/5 and promoting the release of IL-1ß and TNF-α. The levels of epithelial tight junction proteins occludin and claudin-1 decreased. The expression of the pro-apoptotic gene Bax increased. The levels of LPS and TNF-α in systemic circulation increased in hyperuricemic mice. A positive correlation was observed between the increase in intestinal permeability and serum levels of uric acid. CONCLUSION: Hyperuricemia was characterized by dysregulated intestinal immunity, compromised intestinal barrier, and systemic inflammation. These findings might serve as a basis for future novel therapeutic interventions for hyperuricemia.

The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

Comparative Transcriptome Analysis Reveals Candidate Genes Related to Structural and Storage Carbohydrate Biosynthesis in Kelp Saccharina japonica (Laminariales, Phaeophyceae).

Saccharina japonica is a brown macroalga that has been commercially cultivated in China for almost a century. As a natural raw material, it is widely used in the food and pharmaceutical industries, and it may potentially be useful for biofuel production. However, little is known about the genes involved in carbohydrate biosynthesis, and their regulation is less understood. In this study, the analysis of growth traits and alginate and mannitol contents suggested that sporophyte development could be divided into four stages. Accordingly, we performed transcriptome analysis of the S. japonica sporophyte. In total, 589 million clean reads were generated, and 4,514 novel genes were identified. Gene expression analysis revealed that 2,542 genes were differentially expressed. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that these genes were significantly enriched in "Carbon metabolism," "Photosynthesis," and "Photosynthesis-antenna proteins" pathways, which are important for metabolism of various carbohydrates during sporophyte development. Systematic analysis identified the genes encoding enzymes for the biosynthesis of cell wall carbohydrates (including alginate, fucoidan, and cellulose) and cytoplasm storage carbohydrates (mannitol, laminarin, and trehalose). Among them, some key genes associated with carbohydrate content were further identified based on detailed expression profiling, representing good candidates for further functional studies. This study provides a global view of the carbohydrate metabolism process and an important resource for functional genomics studies in S. japonica. The results obtained lay the basis for elucidating the molecular mechanism of carbohydrate biosynthesis and for genetic breeding of carbohydrates-related traits in kelp.

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga.

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

The altered gut microbiota of high-purine-induced hyperuricemia rats and its correlation with hyperuricemia.

Some studies on the hyperuricemia (HUA) have focused on intestinal bacteria. To better understand the correlation between gut microbiota and HUA, we established a HUA rat model with high-purine diet, and used 16S rRNA genes sequencing to analyze gut microbiota changes in HUA rats. To analyze the potential role played by gut microbiota in HUA, we altered the gut microbiota of HUA rats with antibiotics, and compared the degree of uric acid elevation between HUA and antibiotic-fed HUA rats (Ab+HUA). Finally, we established a recipient rat model, in which we transplanted fecal microbiota of HUA and normal rats into recipient rats. Three weeks later, we compared the uric acid content of recipient rats. As a result, the diversity and abundance of the gut microbiota had changed in HUA rats. The Ab-fed HUA rats had significantly lower uric acid content compared to the HUA rats, and gut microbiota from HUA rats increased uric acid content of recipient rats. The genera Vallitalea, Christensenella and Insolitispirillum may associate with HUA. Our findings highlight the association between gut microbiota and HUA, and the potential role played by gut microbiota in HUA. We hope that this finding will promote the isolation and culture of HUA-related bacteria and orient HUA-related studies from being correlational to mechanistic. These steps will therefore make it possible for us to treat HUA using gut microbiota as the target.

Hyperuricemia is associated with impaired intestinal permeability in mice.

Hyperuricemia is associated with many metabolic diseases. However, the underlying mechanism remains unknown. The gut microbiota has been demonstrated to play significant roles in the immunity and metabolism of the host. In the present study, we constructed a hyperuricemic mouse model to investigate whether the metabolic disorder caused by hyperuricemia is related to intestinal dysbiosis. A significantly increased intestinal permeability was detected in hyperuricemic mice. The difference in microflora between wild-type and hyperuricemic mice accompanies the translocation of gut microbiota to the extraintestinal tissues. Such a process is followed by an increase in innate immune system activation. We observed increased LPS and TNF-α levels in the hyperuricemic mice, indicating that hyperuricemic mice were in a state of low-grade systemic inflammation. In addition, hyperuricemic mice presented early injury of parenteral tissue and disordered lipid metabolism. These findings suggest that intestinal dysbiosis due to an impaired intestinal barrier may be the key cause of metabolic disorders in hyperuricemic mice. Our findings should aid in paving a new way of preventing and treating hyperuricemia and its complications.NEW & NOTEWORTHY Hyperuricemia is associated with many metabolic diseases. However, the underlying mechanism remains unknown. We constructed a hyperuricemic mouse model to explore the relationship between intestinal dysbiosis and metabolic disorder caused by hyperuricemia.

Genome assembly of Nannochloropsis oceanica provides evidence of host nucleus overthrow by the symbiont nucleus during speciation.

The species of the genus Nannochloropsis are unique in their maintenance of a nucleus-plastid continuum throughout their cell cycle, non-motility and asexual reproduction. These characteristics should have been endorsed in their gene assemblages (genomes). Here we show that N. oceanica has a genome of 29.3 Mb consisting of 32 pseudochromosomes and containing 7,330 protein-coding genes; and the host nucleus may have been overthrown by an ancient red alga symbiont nucleus during speciation through secondary endosymbiosis. In addition, N. oceanica has lost its flagella and abilities to undergo meiosis and sexual reproduction, and adopted a genome reduction strategy during speciation. We propose that N. oceanica emerged through the active fusion of a host protist and a photosynthesizing ancient red alga and the symbiont nucleus became dominant over the host nucleus while the chloroplast was wrapped by two layers of endoplasmic reticulum. Our findings evidenced an alternative speciation pathway of eukaryotes.

Fabrication of hydroxyapatite/hydrophilic graphene composites and their modulation to cell behavior toward bone reconstruction engineering.

Cell adhesion was the first step of bone reconstruction. While hydroxyapatite (HA)/graphene composites had been utilized for improving the cell adhesion and bone osteogenesis, the impact of cell adhesion and HA/graphene composites, especially HA/hydrophilic graphene (HG) composites, on internal interaction force and external surface properties remained poorly understood. Here, higher stability HA/HG composites were synthesized without extra ion introduction with in situ self-assembling method. And with XRD, FT-IR, XPS and Raman analyses, the evidences of the formation of HA and the introduction of HG was clear. TEM and SEM images showed the net-like spatial structure due to the internal interaction force between HA and HG, which provided the strain stimulation for cell adhesion. Subsequently, the external surface properties of HA/HG composites demonstrated that the roughness and hydrophilic ability of HA/HG composites could be artificially regulated by increasing the content of HG. Besides, the cell proliferation rate of HA/HG composites had been investigated. Compared to the intrinsic HA, HA/5%HG possessed the higher cell proliferation rate (264.81%) and promoted the spreading and growth of MC3T3-E1 cells. Finally, the regulation mechanism between HA/HG and cell adhesion were illuminated in detail. The excellent regular behavior of HA/HG composites for cell adhesion made them promising candidates for bone reconstruction and repairing. The present work provided the reference for the design of modifiable biomaterials and offered much inspiration for the future research of bone reconstruction engineering.

The Role of Malic Enzyme on Promoting Total Lipid and Fatty Acid Production in Phaeodactylum tricornutum.

To verify the function of malic enzyme (ME1), the ME1 gene was endogenously overexpressed in Phaeodactylum tricornutum. Overexpression of ME1 increased neutral and total lipid content and significantly increased saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) in transformants, which varied between 23.19 and 25.32% in SFAs and between 49.02 and 54.04% in PUFAs, respectively. Additionally, increased ME1 activity was accompanied by elevated NADPH content in all three transformants, indicating that increased ME1 activity produced additional NADPH comparing with that of WT. These results indicated that ME1 activity is NADP-dependent and plays an important role in the NADPH levels required for lipid synthesis and fatty acid desaturation in P. tricornutum. Furthermore, our findings suggested that overexpression of endogenous ME1 represents a valid method for boosting neutral-lipid yield in diatom.

Heterogeneity of intron presence/absence in Olifantiella sp. (Bacillariophyta) contributes to the understanding of intron loss.

Although hypotheses have been proposed and developed to interpret the origins and functions of introns, substantial controversies remain about the mechanism of intron evolution. The availability of introns in the intermediate state is quite helpful for resolving this debate. In this study, a new strain of diatom (denominated as DB21-1) was isolated and identified as Olifantiella sp., which possesses multiple types of 18S rDNAs (obtained from genomic DNA; lengths ranged from 2,056 bp to 2,988 bp). Based on alignments between 18S rDNAs and 18S rRNA (obtained from cDNA; 1,783 bp), seven intron insertion sites (IISs) located in the 18S rDNA were identified, each of which displayed the polymorphism of intron presence/absence. Specific primers around each IIS were designed to amplify the introns and the results indicated that introns in the same IIS varied in lengths, while terminal sequences were conserved. Our study showed that the process of intron loss happens via a series of successive steps, and each step could derive corresponding introns under intermediate states. Moreover, these results indicate that the mechanism of genomic deletion that occurs at DNA level can also lead to exact intron loss.