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.

不同电荷修饰的聚苯乙烯纳米塑料对海洋微拟球藻(Nannochloropsis oceanica)生长及光合活性的影响

评估了30 nm阴离子羧基修饰(-COOH)和100 nm阳离子氨基修饰(-NH_(2))的聚苯乙烯(PS)NPs及其不同浓度下(PS-COOH:0、2、5、8和10 mg·L^(-1);PS-NH_(2):0、0.2、0.3、0.4和0.5 mg·L^(-1))对海洋微藻微拟球藻(Nannochloropsis oceanica)在生长及光合活性中产生的影响。结果表明,两种NPs在培养基中均出现了聚集和电位绝对值的下降;经过羧基修饰的PS对海洋微拟球藻的生长及光合活性并未产生明显的影响,而经过氨基修饰的PS在不同浓度下对海洋微拟球藻的生长及光合活性产生了不同的影响:低浓度(0.2 mg·L^(-1))PS-NH_(2)对海洋微拟球藻生长产生促进作用,高浓度(0.4和0.5 mg·L^(-1))PS-NH_(2)对海洋微拟球藻生长产生抑制作用,海洋微拟球藻在不同浓度PS-NH_(2)胁迫下显示出了低促高抑现象;并且当海洋微拟球藻暴露于0.2 mg·L^(-1)PS-NH_(2)时,叶绿素a(Chl a)含量较对照组高,而其他处理组均低于对照组;此外,在0.2 mg·L^(-1)PS-NH_(2)处理下的海洋微拟球藻Fv/Fm值一直高于对照组,而在0.5 mg·L^(-1)高浓度的PS-NH_(2)胁迫下,海洋微拟球藻的最大光化学效率始终低于对照组,这些结果与生长数据相吻合。

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.

Assessment of <i>Posidonia Oceanica</i>(L.) Delile conservation status by standard and putative approaches: the case study of Santa Marinella meadow (Italy, W Mediterranean)

The conservation status of the Posidonia oceanica meadow at Santa Marinella (Rome) was evaluated through both standard (bed density, leaf biometry, "A" coefficient, Leaf Area Index, rhizome production) and biochemical/genetic approaches (total phenol content and Random Amplified Polymorphic DNA marker). The bio-chemical/genetic results are in agreement with those obtained by standard approaches. The bed under study was ranked as a disturbed one, due to its low density, and high heterogeneity in leaf biometry, LAI values, "A" coefficient and primary production. This low quality ranking is confirmed by both mean phenol content in plants, quite high and scattered, and by the low genetic variability in the meadow, with a very high similarity of specimen at a local scale. Hence, these two putative approaches clearly identify the endangered conservation status of the meadow. They link plant biodiversity and ecophysiology to ecosystem 'health'. Furthermore, they are repeatable and standardizable and could be usefully introduced in meadows monitoring to check environmental quality.

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.

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.

Trace metals in <i>Posidonia oceanica</i>in a coastal area of the Ionian Sea (Calabria, Italy)

As, Cd, Cu and Pb concentrations were measured in Posidonia oceanica sampled from meadows located in two sites along the calabrian coast (Ionian Sea, South-eastern Mediterranean). By dating the scales and the rhizome using retrospective procedures (lepidochronology), a time series over a period of nine years (1995-2004) was analysed. Throughout the whole lepidochronological period, the arsenic content in the scales was tenfold higher than that measured in the rhizome with a peak in the lepidochronological year 1996-1997 and a general decrease in the years after. Pb concentration also showed a tendency to decrease with time, whereas the Cd and Cu concentration were increasing. A comparison of the metal level in dead sheaths (scales) and living tissue (leaves) was also performed. The detailed distribution of the trace metals along the leaf axis and in the leaves at different developmental stages (adult, intermediate and juvenile) showed some differences in the metal content. The results also indicate that the arsenic content measured in the leaf blade reflects the high As content measured at the superficial sediments at one of the two sampled sites, demonstrating, for the first time, that P. oceanica would be a good indicator for this element.

Epiphytic Diatom Communities on Sub-Fossil Leaves of <i>Posidonia oceanica</i>Delile in the Graeco-Roman Harbor of Neapolis: A Tool to Explore the Past

The Graeco-Roman harbor of Neapolis (Naples, Italy), chronologically constrained between the late 4th century BC and the 6th century AD, offers a unique sight on relationship between past and present into the history of the marine biodiversity. In fact, the digs expose fossil leaf of Posidonia oceanica with epiphytic communities of microorganisms. Posidonia oceanica is a seagrass endemic to theMediterraneanthat forms large meadows whose remains can persist in the sediment for thousands of years. In this communication, we report results of analyses carried out using both molecular and morphological techniques on sub-fossil leaves of P. oceanica and their associated epiphytic communities.

Comparative analysis of bed density, total phenol content and protein expression pattern in <i>Posidonia oceanica</i>(L.) Delile

Posidonia oceanicameadows are experiencing a progressive decline, and monitoring their status is crucial for the maintenance of theseecosystems. We performed a comparativeanalysis of bed density, total phenol content and protein expression pattern to assess the conservation status ofPosidoniaplants from the S. Marinella (Rome, Italy) meadow. The total phenol content was inversely related to maximum beddensity, confirming the relationship betweenhigh phenol content and stressful conditions. In addition, protein expression pattern profilesshowed that the number of differentially expressed proteins was dramatically reduced in the latest years compared to previous analyses. Our results support the usefulness of integrating solid descriptors, such as phenol content, with novel biochemical/molecular approaches in the monitoring of meadows.

Effect of Nitrogen Source on Biomass and Lipid Production of a Marine Microalga, <i>Nannochloropsis oceanica</i>IMET1

The effects of the nitrogen sources sodium nitrate (NaNO3) and urea (CH4N2O) on growth, lipid production, and fatty acid composition of Nannochloropsis oceanica IMET1 were investigated. Nitrogen source affected cell density, dry cell weight, and lipid production. Cells grown in the nitrate medium increased dry cell weight and lipid weight in comparison with cells grown in the urea medium. The composition of fatty acids varied with nitrogen sources. IMET1cultured in the nitrate medium mainly contained C18:2 (14.9%) and C16:0 (6.3%) fatty acids, while IMET1 in the urea medium mainly contained C22:0 (33.1%), C18:3 (8.6%), and C16:0 (6.8%). This study demonstrates that nitrogen source can strongly influence lipid production and composition of N. oceanica IMET1.

Diet of fish populations in posidonia oceanica meadows off the Island of Ischia (Gulf of Naples, Italy):

The gut contents of fish in three Posidonia oceanica meadows off the island of Ischia (Bay of Naples, Italy) were investigated. A total of 926 individual fish belonging to 28 species was sampled by bottom trawl in the leaf canopy. Labridae, Pomacentridae, Scorpaenidae, and Serranidae were the best represented families (41%, 38%, 8% and 6% of the total number of individuals, respectively). Of the 94 taxa detected in the gut contents, 42 were identified to the species level. The most common food items were decapod crustaceans (15% of the gut contents, on average), copepods (13%), amphipods (14%), brown fragments of P. oceanica (6%), and ostracods (6%). The most abundant species of labridae, Symphodus ocellatus and S. rostratus, showed a broad spectrum of prey. This generalist feeding may positively influence their numerical abundance. Seasonal variations in the diets of fish, also at prey-species level, were demonstrated. The fish taxon plays essentially a macro-carnivore trophic role. In the investigated seagrass meadows the main trophic fluxes start from plant detritus, macrophyta, and microphyta (as primary producers) towards crustacean decapods, copepods, ostracods, and gammarid amphipods (as secondary producers) to fish. A low recycling rate (4%) within the fish community was observed. Larger fish predators (e.g., Sparidae), swimming over the leaf canopy, are the main exporters to adjacent coastal systems.

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.

Restoration of Posidonia oceanica （L.） Delile Meadows： Is There An Effective Methodology？

Since 1947 （Addy） [1, 2], marine biologists have conducted experiments in order to re-establish meadows using various methods of transplant. A 27-month experiment of germinating and growing Posidonia oceanica （L.） Delile seeds was carried out in two areas near Piombino in the Leghorn Province （Italy）. Seeds in direct contact with the sea bottom were taken from other seaside areas. The leaf growth and the percentage of well-developed plants in the study areas were calculated. The results confirm that this method appears to be a good re-establishment technique despite the problems associated with finding a large number of seeds anywhere along Mediterranean coasts.

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.

Variability in patterns of macro-epiphytic leaf community of Posidonia oceanica in the Islands of Kuriate:Western coast of Tunisia

Objective:To test the response of the epiphyte community structure and biomass of the Posidonia oceanica(P.oceanica)leaves to natural disturbance.Methods:Sampling of P.oceanica was carried in winter and summer on three sites in Kuriate Islands(western coast of Tunisia)subject to different environments disturbances.Shoots of P.oceanica were preserved in seawater-formalin(5%)solution for macro-epiphytes species identification in the laboratory.The samples were examined for leaf surface per shoot and the coverage(expressed as a percentage of leaf surface)of each morphological group,then carefully scraped with a razor blade.Epiphytes and scraped leaves were oven-dried at 60℃for 48 h.Biomass was expressed as g dry weight/shoot.Results:The biomass and the percentage cover of macro-epiphytic leaves showed seasonal variation.The highest values of epiphytic leaves were detected in summer whereas the lowest values were registered during winter.ANOVA showed that Kuriate Islands functioned as a single ecosystem in terms assemblage of macro-epiphytic leaves since no significant variation was detected for biomass and percentage cover at the scale site.Our study showed that natural disturbance had no effect on the assemblage distribution and the biomass of macro-epiphyte on the leaves of P.oceanica between the scales of site,whereas variability at the smallest scale was detected.ANOVA showed that exposure to wind and current had no effect on the biomass of macro-epiphytes leaves.Conclusions:Biomass and assemblages of macro-epiphytic leaves of P.oceanica were high in summer and homogenous between all sites investigated.Natural disturbances such as exposure to wind have no effect on the distribution and the biomass of epiphytes on the shallow meadow.

酶菌协同处理对微拟球藻营养功能成分及生物活性的影响

为提高微拟球藻开发利用价值,本文开展了酶菌协同处理对微拟球藻营养功能成分及生物活性影响的研究。采用木瓜蛋白酶酶解微拟球藻,利用单因素和响应面试验获得其最佳酶解工艺,随后通过益生菌发酵酶解基质,研究发酵前后总酚、多肽和黄酮含量以及体外抗氧化和降血脂活性的变化,并对发酵后的微拟球藻酶液进行感官评价。结果表明:微拟球藻的最佳酶解工艺为:酶量5.0%、pH8.0、酶解时间4.0 h、酶解温度76℃;在此条件下酶解后发酵可有效提高产物中多肽的含量,其中混菌发酵组提高了近1.3倍,总酚含量提高了5.1%,同时总黄酮的含量降低了32.5%;酶菌协同处理显著增强了其抗氧化和胰脂肪酶抑制活性,其中混菌发酵组DPPH自由基清除率提高了1.2倍,胰脂肪酶清除率提高了59.4%。此外,发酵后感官评分显著提升。可见,酶菌协同发酵可显著提高微拟球藻多肽含量、抗氧化活性、降血脂活性及感官特性,为开发功能型发酵产品提供了参考和技术支持。