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.

Nannochloropsis artificial chromosomes(Nanno ACs)loom on the horizon

Species in genus Nannochloropsis,especially N.oceanica and N.gaditana,have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration,the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation,homologous recombination,gene edition,gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with“top down”strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes(NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review,we outlined the current status of transgenes in Nannochloropsis species,summarized the limitations of both integrative and non-integrative vectors,and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first,and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds,thus contributing towards its genetic improvement and synthetic biological studies.

通气量和CO_2对Nannochloropsis sp.在光生物反应器中的生长和EPA合成的影响

实验考察了在气升式内环流光生物反应器中通气量、CO2含量等培养条件对Nannochloropsissp.生长及EPA合成的影响.结果表明,在气升式内环流光生物反应器中培养,Nannochloropsissp.生长速率显著提高.培养8d,Nannochloropsissp.生物量(干重)可达857mg/L,是摇床培养的2倍.在一定范围内,Nannochloropsissp.的生长速率随通气量的增加而增加,在本实验条件下,通气量为500mL/min时生长最快,而过高的通气量则对Nannochloropsissp.的生长没有促进作用.在通气中含1%(?)CO2时,可加快藻细胞的生长,最大生长速率可达不配加CO2时的1.8倍.通气量和CO2对Nannochloropsissp.细胞内总脂肪酸及EPA的积累有显著影响.在通气量为400mL/min及CO2含量为0.5%时,培养液中EPA产量最高,达到39.0mg/L.

CO_2对微拟球藻(Nannochloropsis sp.)利用有机碳和光合作用的影响

CO2浓度提高时,微拟球藻吸收醋酸钠的速率增加2倍。混养生长的藻细胞最大光合作用速率、光合作用效率、无机碳半饱和常数和无机碳饱和的光合作用速率均显著低于光自养条件下生长的。

Effects of L-carnitine on Population Growth of Nannochloropsis oculata and Tetrahymena sp.

[Objective] The aim was to study the effects of L-carnitine on population growth of Nannochloropsis oculata and Tetrahymena sp..[Method] When the concentration of L-carnitine was 0,50,100 and 1 000 mg/L,population densities of Nannochloropsis oculata and Tetrahymena were determined respectively.[Result] Adding high-dose L-carnitine had a significant inhibition effect on the population growth of Nannochloropsis oculata （P0.05）.Adding L-carnitine had a significant proliferation promoting effect on the population growth of Tetrahymena （P0.05）.[Conclusion] The research provides theoretical basis for the application of L-carnitine as feed additive in aquaculture.

海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5的转化及外源基因整合方式

微拟球藻Nannochloropsis被认为是具有作为生物柴油原料开发潜力的微藻。为了能够实现工业化生产,有效地利用基因工程或遗传操作手段改造微藻,提高产量,建立稳定有效的遗传转移方法十分必要。本研究以微拟球藻本源β-tublin基因启动子和三角褐指藻Phaeodactylum tricornutm fcpA终止子驱动和终止来源于细菌的sh ble抗性选择基因,构建了一个转化载体pHB4857。将pHB4857以电转移的方法转化海洋富油微拟球藻Nannochloropsis gaditana CCAP849/5。结果显示,转化子可以在3μg·mL-1 zeocin的抗性培养基中生长,PCR检测sh ble基因为100%插入率,转化效率为1.25×10-6。DNA印迹杂交结果表明,外源基因是以随机整合的方式一个或多个拷贝插入到宿主核基因组中的,大多数转化子中的外源基因的整合是完整的。转化子在抗性培养基中每10天传一代,连续传代7个月以上,未检测到抗性基因丢失现象,外源抗性基因可以在宿主细胞中稳定存在。

Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

A gene （NANOC-D6D） encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata （Droop） D. J. Hibberd （Eustigmatophyceae）. The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids （LCPUFAs）, including eicosapentaenoic acid （20：5n-3, EPA）. The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NAN OC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid （LA） and a-linolenic acid （ALA）.

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.

Cloning and Phylogenetic Analysis of a Fatty Acid Elongase Gene from Nannochloropsis oculata CS179

Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids （LCPUFAs）. Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5＇ and 3＇ RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the coptosporidium. But the NJ-tree revealed that the N. oculata CS 179 elongase clustered with those of the microalgae Phaeodac^lum tricornutum, Ostreocoecus tauri and Thalassiosira pseudonana.

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.

Construction and characterization of a normalized cDNA library of Nannochloropsis oculata(Eustigmatophyceae)

We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179,and obtained 905 nonredundant sequences(NRSs) ranging from 431-1 756 bp in length.Among them,496 were very similar to nonredundant ones in the GenBank(E ≤1.0e-05),and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups(KOG).Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs(>60%).We also identified the unigenes encoding phosphorus and nitrogen transporters,suggesting that N.oculata could efficiently transport and metabolize phosphorus and nitrogen,and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids(PUFAs),which will facilitate the demonstration of eicosapentaenoic acid(EPA) biosynthesis pathway of N.oculata.In comparison with the original cDNA library,the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering,and decreased the frequency of abundant gene sequences.

Low-Temperature Affected LC-PUFA Conversion and Associated Gene Transcript Level in Nannochloropsis oculata CS-179

Nannochloropsis oculata CS-179, a marine eukaryotic unicellular microalga, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs). Culture temperature affected cell growth and the composition of LC-PUFAs. At an initial cell density of 1.5 × 106 cell mL-1, the highest growth was observed at 25℃ and the cell density reached 3 × 107 cell mL-1 at the beginning of loga-rithmic phase. The content of LC-PUFAs varied with culture temperature.The highest content of LC-PUFAs (43.96%) and EPA (36.6%) was gained at 20℃. Real-time PCR showed that the abundance of Δ6-desaturase gene transcripts was significantly different among 5 culture temperatures and the highest transcript level (15℃) of Nanoc-D6D took off at cycle 21.45. The gene transcript of C20-elongase gene was higher at lower temperatures (10, 15, and 20℃), and the highest transcript level (20℃) of Nanoc-E took off at cycle 21.18. The highest conversion rate (39.3%) of Δ6-desaturase was also gained at 20℃.But the conversion rate of Nanoc-E was not detected. The higher content of LC-PUFAs was a result of higher gene transcript level and higher enzyme activity. Compared with C20-elongase gene, Δ6-desaturase gene transcript and enzyme activity varied significantly with temperature. It will be useful to study the mechanism of how the content of LC-PUFAs is affected by temperature.

Identification of a Long-Chain Fatty Acid Elongase from Nannochloropsis sp. Involved in the Biosynthesis of Fatty Acids by Heterologous Expression in Saccharomyces cerevisiae

The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.

Temperature effects on lipid properties of microalgae Tetraselmis subcordiformis and Nannochloropsis oculata as biofuel resources

Microalgae Tetraselmis subcordiformis and Nannochloropsis oculata were cultured at 15,20,25,30,and 35℃ and their properties as potential biofuel resources were examined.The results indicate that T.subcordiformis and N.oculata grew best at 20℃ and 25℃ and yielded the highest total lipids at 20℃and 30℃,respectively.With increased temperature,neutral lipid and polyunsaturated fatty acids(FAs)decreased while saturated FAs increased,accompanied by increased monounsaturated FAs(MUFAs) in T.subcordiformis and decreased MUFAs in N.oculata;meanwhile,the predicted cetane number of FA methyl esters increased from 45.3 to 47.6 in T.subcordiformis and from 52.3 to 60.3 in N.oculata.Therefore,optimizing culture temperatures is important for improving microalgal biodiesel production.

Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated (~730 generations,~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colony-derived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed;(2) the physiological processes in which these genes involved have been modified;and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.

Transcriptomic Response of Nannochloropsis oceanica to Benzo[a]pyrene

In this study, the RNA sequencing was used to describe the response of Nannochloropsis oceanica, a marine microalga, to benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbon, in order to elucidate the metabolic pathways(or processes) involved in microalgal response to this stubborn pollutant. N. oceanica was exposed to BaP at a concentration of 90 μg L^-1 for 72 h, and its transcriptome was sequenced through the Illumina HiSeq^TM 2500 platform. This concentration of BaP was selected as it is the lowest for modeling the most appropriate growth inhibition of N. oceanica for transcriptomic analysis. We found that N. oceanica responds to BaP through degrading proteins and repairing DNA damaged by BaP. In addition, superoxide dismutase(SOD) strengthened its performance by increasing its transcript abundance. The physiological mechanism underlining the response of N. oceanica to BaP as revealed by transcriptomic analysis was consistent with the biochemical insights documented previously.

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

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.