An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Verification of Mutagen Function of Zeocin in Nannochloropsis oceanica Through Transcriptome Analysis

Zeocin can cause double strand breaks of DNA and thus is frequently used as a selective antibiotic of eukaryotic Sh ble transformants. In non-transformation system, Zeocin may function as a mutagen if not totally lethal. To verify such function of Zeocin, we mutated Nannochloropsis oceanica by increasing the concentration of Zeocin in medium gradually, and isolated a N. oceanica strain(single cell culture) which survived Zeocin up to 10.0μg mL^(-1). The Zeocin-tolerant strain entered the exponential growth phase later and grew slower than the wild strain. Transcriptome profiling showed that the Zeocin-tolerant N. oceanica strain survived Zeocin mainly by adapting(heritable), rather than acclimating(plastic) to Zeocin. Hence mutating N. oceanica with Zeocin was approved effective. Meanwhile, the physiological characteristics of this Zeocin-tolerant strain were demonstrated. As we proposed, N. oceanica tolerated Zeocin by strengthening its protein degradation and antioxidation. The genes controlling cell division and cellular response to stimuli may also have played important roles in the reduction of growth and the tolerance to Zeocin. Our findings evidenced that Zeocin can serve as an appropriate mutagen of microalgae. Creating variations through mutation with Zeocin may help to study the genetic basis of the traits of this monoploidy and asexual microalga, as well as improve its production.

Improvement of Nannochloropsis oceanica growth performance through chemical mutation and characterization of fast growth physiology by transcriptome profiling

Nannochloropsis oceanica promises to be an industrial-level producer of polyunsaturated fatty acids. In this study, the fastest and slowest growing N. oceanica mutants were selected through N-methyl-N＇-nitro-N-nitrosoguanidine mutation, and two mutant strains and the wild type （WT） subjected to transcriptome profiling. It was found that the 0D680 reads at stationary growth phase of both WT and its mutants were proportional to their cell density, thus indicating their division rate and growth speed during culture. This chemical mutation was effective for improving growth performance, and the fast strain divided faster by upregulating the expression of genes functioning in the cell cycle and downregulating genes involved in synthesis of amino acids, fatty acids, and sugars as well as the construction of ribosome and photosynthetic machinery. However, the relationship among the effected genes responsible for cell cycle, metabolism of fatty and amino acids, and construction of ribosome and photosynthetic machinery remained unclear. Further genetic studies are required for clarifying the genetic/metabolic networks underpinning the growth performance ofN. oceanica. These findings demonstrated that this mutation strategy was effective for improving the growth performance of this species and explored a means ofmicroalgal genetic improvement, particularly in species possessing a monoploid nucleus and asexual reproduction.

The Mechanism of the Acclimation of Nannochloropsis oceanica to Freshwater Deduced from Its Transcriptome Profiles

In this study, we compared the transcriptomes of Nannochloropsis oceanica cultured in f/2 medium prepared with seawater and freshwater, respectively, aiming to understand the acclimation mechanism of this alga to freshwater. Differentially expressed genes were mainly assigned to the degradation of cell components, ion transportation, and ribosomal biogenesis. These find- ings indicate that the algal cells degrade its components （mainly amino acids and fatty acids） to yield excessive energy （ATP） to maintain cellular ion （mainly K＋ and Ca〉） homeostasis, while the depletion of amino acids and ATP, and the reduction of ribosomes attenuate the protein translation and finally slow down the cell growth.

Structural Variation Analysis of Mutated Nannochloropsis oceanica Caused by Zeocin Through Genome Re-Sequencing

Zeocin can cause double strand breaks of DNA and thus may be employed as a mutagen. In this study, two strains of Nannochloropsis oceanica, the wild and the Zeocin-tolerant strains, were re-sequenced to verify such function of Zeocin, The results showed that Zeocin can mutate the N. oceanica genome and cause the structural variation. Zeocin either swept away or selected the alleles of genes functioning in ubiquitin-mediated proteolysis, alpha-linolenic acid metabolism, ascorbate and aldarate metabolism, ribosome biogenesis, and circadian rhythm, indicating that N. oceanica may have adjusted its metabolic performances for protein, carbohydrate, and lipid, and changed its ribosome biosynthesis and living rhythm to survive in Zeocin containing medium. In addition, Zeocin caused mutation may have influenced the expression of a set of tanscription factors. It was concluded that Zeocin effectively caused the structural variation of the genome of N. oceanica, and forced the microalgae to select out the alleles of a set of genes around these variations in order to adapt to Zeocin containing medium. Further studies on the genetic basis of the phenotypic adaptation of this haploid and asexual microalga and the application of Zeocin to its genetic improvement are very important.

Transcriptome Responses of Hygromycin B Resistance Gene-Transformed, Hygromycin B-Adaptive and Wild Nannochloropsis oceanica Strains to Hygromycin B

In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica,the transcriptome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene,a hygromycin B-adaptive strain and a wild type strain of N.oceanica were compared by transcriptome sequencing(RNA-seq)without referring to a high quality genome sequence.The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes,thus reducing the intracellular concentration of hygromycin B.The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene.Accordingly,the screening and maintenance of N.oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold.Our findings can help the genetic modification of N.oceanica with the application of hygromycin B.

Digging out molecular markers associated with low salinity tolerance of Nannochloropsis oceanica through bulked mutant analysis

Nannochloropsis oceanica is a marine microalgal species with both economic value and biological importance.It grows fast,contains rich oils,reproduces asexually,holds a small and haploidy genome,and is easy to be modified genetically.However,the genetic study of N.oceanica is scarce.Very less genetic bases of its traits have been deciphered,and no gene has been isolated from it with the function verified simultaneously via either genetic or reverse genetic approaches or both(de novo cloned).Changing medium salinity may aid to control harmful organisms met during large scale cultivation.As a stress,it may also facilitate the accumulation of desirable chemicals including fatty acids.In order to decipher the genetic basis of the low salinity tolerance of N.oceanica,we mutated N.oceanica with Zeocin.In total,five mutant bulks were constructed at equal number of cells,100 mutants each,which were tolerant to a discontinuous serial of salinities from that of 100%of f/2 to that of a mixture of 4%of f/2 and 94%of BG11.The bulks were genotyped through whole genome re-sequencing and analyzed with bulked mutant analysis(BMA)newly modified from bulked segregant analysis(BSA).In total,47 SNPs and 112 InDels were found to associate with the low salinity tolerance,and around them a set of low salinity tolerance associating genes were identified.A set of annotatable genes commonly found between control and different salinities indicated that the genes functioning in gene expression,energy metabolism and cellular structure may be involved in the low salinity tolerance.These associating molecular markers and genes around them were not enough for outlining the physiological mechanism underlining the tolerance;however they should aid to improve N.oceanica genetically.

An investigation of the possible methods and potential benefits of de novo cloning of Nannochloropsis oceanica genes

Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Herewe attempt to generate InDei variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basisof its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, wedescribe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse geneticapproaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, nofunctional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatablegenes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.

Molecular Identification of a Species in Genus Nannochloropsis

Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA,such as DNA methylation at N6-adenine(6mA),play crucial roles in gene expression and genome maintenance,but their deposition and function in microalgae remain largely uncharacterized.Here,we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing.Found in 0.1%of adenines,6mA sites are mostly enriched at the AGGYV motif,more abundant in transposons and 30 untranslated re-gions,and associated with active transcription.Moreover,6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcrip-tion termination sites.Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes,indicating a positive interaction between 6mA and general transcription fac-tors.Furthermore,knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor,sulfate transporter,glycosyl transferase,and lipase genes that underlie reductions in biomass and oil productivity.By contrast,knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth.Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.