Identification of novel salt stress-responsive microRNAs through sequencing and bioinformatic analysis in a unique halophilic Dunaliella salina strain

Dunaliella salina is a classic halophilic alga.However,its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown.A unique halophilic alga strain,DS-CN1,was screened from four D.salina strains via cell biological,physiological,and biochemical methods.High-throughput sequencing of small RNAs(sRNAs)of DS-CN1 in culture medium containing 3.42-mol/L NaCl(SS group)or 0.05-mol/L NaCl(CO group)was performed on the BGISEQ-500 platform.The annotation and sequences of D.salina sRNAs were profiled.Altogether,44 novel salt stress-responsive microRNAs(miRNAs)with a relatively high C content,with the majority of them being 24 nt in length,were identified and characterized in DS-CN1.Twenty-one differentially expressed miRNAs(DEMs)in SS and CO were screened via bioinformatic analysis.A total of 319 putative salt stress-related genes targeted(104 overlapping genes)by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research.Furthermore,these target genes were classified and enriched by GO and KEGG pathway analysis.Moreover,5 novel DEMs(dsa-mir3,dsa-mir16,dsa-mir17,and dsa-mir26 were significantly upregulated,and dsa-mir40 was significantly downregulated)and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR.Next,their regulatory relationships were comprehensively analyzed.Lastly,a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs.Taken together,our results suggest that 44 novel salt stress-responsive microRNAs were identified,and 4 of them might play important roles in D.salina upon salinity stress and contribute to clarify its distinctive halophilic feature.Our study will shed light on the regulatory mechanisms of salt stress responses.

Fe对两株盐生杜氏藻(Dunaliella salina)生长和β-胡萝卜素积累的影响

研究了柠檬酸铁对两株盐生杜氏藻Dunaliella salina OUN04和D.salina OUN09生长和色素积累的影响,结果表明,D.salina OUN04生长最适的Fe浓度为0.05mmol/L,细胞密度达111.5×104cell/mL,0.25mmol/LFe组最低(70×104cell/mL);最大β-胡萝卜素含量(83.2mg/g)出现在0.25mmol/LFe浓度组中;Fe浓度为0.25mmol/L时有最大的叶绿素a含量(98.4mg/g);建立了杜氏藻对Fe吸收的动力学方程。D.salina OUN09生长最适的Fe浓度为0.05mmol/L(密度131×104cell/mL),最大β-胡萝卜素含量为130.2mg/g(0.05mmol/LFe组),对照组仅为70.4mg/g。

Dunalidlla bardawil中类胡萝卜素的高效液相色谱分析及其与Dunaliella salina的比较

采用Bood-Pak C18和Nova-Pak C18两种色谱柱对Dunaliella bardewil中的类胡萝卜素进行了高效液相色谱分析和比较,结果表明在相同的实验条件下,Bood-Pak C18对盐藻中类胡萝卜素的分离效果明显好于Nova-Pak C18。对Bond-Pak C18分析结果进行了定性分析,初步确定Dunaliella bardawil含有的类胡萝卜素有14种以上。同时进行了Dunaliella bardewil和Dunaliella salina类胡萝卜素的比较,表明两种藻主要的类胡萝卜素的成分和含量非常接近,色谱图相似程度很高。

杜氏盐藻(Dunaliella salina)超微结构研究

Ｄｕｎａｌｉｅｌｌａ ｓａｌｉｎａ具有薄而不均匀的外被膜。鞭毛为“９＋２”结构。眼点位于细胞质中。内质网丰富。线粒体形状多样。叶绿体呈“杯形”。首次在杜氏藻中发现两个大型造粉粒。细胞核位于叶绿体凹窝中，核仁存在时期很短。

通过cDNARDA法分离和识别盐藻(Dunaliella salina)盐胁迫相关基因

采用cDNA代表性差异分析 (RDA)技术 ,对盐藻在盐胁迫时差异表达的基因进行了分离鉴定 .在分离到的 10个基因中 ,有 5个与已知基因同源 (包括叶绿素a b结合蛋白基因、蛋白磷酸酶I催化亚基基因和 3个核糖体蛋白基因 ) ,还有 5个未知功能基因则是首次在盐藻中被分离 .值得注意的是 ,所有这 5个已知基因的功能都与细胞分裂或盐胁迫有关 .结果表明 :取样时盐藻细胞仍处于恢复阶段 ,所分离到的基因对于盐藻耐盐可能具有重要意义 ;蛋白磷酸酶I的下调表达可能是盐藻调节离子平衡的一个重要过程和细胞分裂受阻的原因所在 ;盐藻减缓细胞分裂速度可能是为了减少能量消耗 ,以留出足够的能量来应对盐胁迫 ;其它 5个未知基因可能也与盐藻适应盐胁迫机制有关 .

Cr^(3+)对盐藻(Dunaliella salina)生长及营养品质的影响

以盐藻Dunaliella salina为材料,设定0ìg·L^(-1)、3ìg·L^(-1)、12ìg·L^(-1)、50ìg·L^(-1)、200ìg·L^(-1)和800ìg·L^(-1)6个添加Cr^(3+)浓度处理,分析测定了不同铬浓度下盐藻的生物量(细胞密度)。蛋白质、a-胡萝卜素和可溶性糖含量。研究结果表明,中低量添加 Cr^(3+)对盐藻的生长有一定的促进作用,在50ìg·L^(-1)和200ìg·L^(-1)Cr^(3+)条件下,盐藻的生物量高于对照,中低量添加Cr^(3+)对盐藻的生长有一定的促进作用,盐藻蛋白质含量比对照分别提高3.06％和 6.55％,Cr^(3+)浓度在200ìg·L^(-1)时,盐藻的a-胡萝卜素和可溶性糖含量比对照分别提高3.93％和2.38％,适当添加Cr^(3+)可提高盐藻蛋白质、a-胡萝卜素和可溶性糖含量,有效改善盐藻的营养品质。

盐生杜氏藻(Dunaliella salina)cDNA文库构建及功能基因筛选(英文)

采用Qiagen公司的植物总RNA提取技术、Clontech公司的CreatorTM技术平台以及SMARTTM技术进行cDNA文库构建。从杜氏藻中提取出了高质量的总RNA,通过PowerScript反转录酶反转录杜氏藻的总RNA,采用LD-PCR、酶处理等方法对cDNA进行等比例扩增、纯化,同时使用CHROMASPIN-400柱子将cDNA分段化,最后将长片段连入pDNR-LIB质粒,1.5kV,25μF电转化大肠杆菌JM109,得到含1.5×106个克隆子的原始文库,滴度为1.5×106cfuml-1。结合酶切和PCR,对该文库的质量进行了鉴定和统计,文库的平均片段插入长度为1.5kb。采用烯醇酶和UDP葡萄糖脱氢酶的EST作为同源探针,对文库中的功能基因进行筛选,并采用放射性原位杂交法,对扩增文库进行了初筛和复筛,得到了含这两条基因全编码序列的cDNA,烯醇酶为1.8kb,UDP葡萄糖脱氢酶为1.9kb,为今后对该种进行大规模功能基因组学研究奠定基础。

利用 RAPD分析杜氏藻属 (Dunaliella)嗜盐种间遗传多样性

本研究首次运用随机引物对杜氏藻属 (Dunaliella)中 6个嗜盐种的基因组 DNA进行RAPD分析。筛选获得的 6个有效引物共扩增出 98个可重复的 DNA片段 ,其中 95条带具有多态性 ,多态性条带的频率为 96 .9%。根据系统进化树图将杜氏藻属 6个种分别归于亲缘关系相对较远的 2个类群中 ,且

杜氏藻Dunaliella peircei超微结构研究

两类糖蛋白纤维构成了Dunaliella peirces的细胞外被,鞭毛和基本分别为“9+2”和“9+0”结构,首次发现的中心粒由9个三联体组成,高尔基体1～2个,眼点位于细胞质中,由二排椭圆形小球构成,内质网丰富,线粒体形状多样,在“杯形”叶绿体中发现了雏形的基粒及“杯”上的小孔,叶绿体前端凹凸不平,后端具大型淀粉核一个,细胞核位于“杯形”叶绿体的凹窝中。

杜氏藻(Dunaliella sp．)多糖DPS-1的提取分离纯化和糖基组成分析

以实验室培养的新鲜的杜氏藻为原料,采用冻结-融化冷水提取、乙醇沉淀,Sephadex G-100柱层析等一系列步骤,分离纯化得到杜氏藻多糖,简称DPS-1。醋酸纤维素薄膜电泳和Sephadex G-50柱层析对DPS-1进行纯度鉴定,结果表明该糖组分均一。IR、UV等理化性质测定及HPAEC-PAD分析,结果表明该糖含有硫酸根,糖环为吡喃环。组成DPS-1的糖基有:D-岩藻糖、D-鼠李糖、2-D-氨基葡萄糖、D-半乳糖、D-葡萄糖和两种未知单糖。其摩尔比为D-岩藻糖∶D-鼠李糖∶2-D-氨基葡萄糖∶D-半乳糖∶D-葡萄糖=2.1∶1∶1.6∶2.3∶4.2。另外还检出了两种已知糖醛酸,分别是半乳糖醛酸和葡萄糖醛酸。其摩尔比为半乳糖醛酸∶葡萄糖醛酸=1∶3.4。

Transcriptome analysis of carotenoid biosynthesis in Dunaliella salina under red and blue light

The quality of light is an important abiotic factor that affects the growth and development of photosynthetic organisms.In this study,we exposed the unicellular green alga Dunaliella salina to red(660 nm)and blue(450 nm)light and analyzed the cell growth,total carotenoid content,and transcriptomes.The growth of D.salina was enhanced by illumination with red light,whereas blue light was not able to promote the algal growth.In contrast,the total carotenoid content increased under both red and blue light.The RNA of D.salina was sequenced and the transcriptomic response of algal cells to red and blue light was investigated.Six transcripts encoding for the blue light receptor cryptochrome were identified,and transcripts involved in the carotenoid metabolism were up-regulated under both red and blue light.Transcripts encoding for photoprotective enzymes related to the scavenging of reactive oxygen species were up-regulated under blue light.The present transcriptomic study provides a more comprehensive understanding of carotenoid biosynthesis in D.salina under different wavelengths of light.

Phylogenetic and Morphological Investigation of a Dunaliella Strain Isolated from Yuncheng Salt Lake,China

A Dunaliella strain was isolated from Yuncheng Salt Lake,Shanxi,China(111.05°E,35.03°N).Morphological characteristics and molecular data were used to evaluate the relationship of this algal strain to other Dunaliella

人canstatin基因转化新型生物反应器——杜氏盐藻(Dunaliella salina)的初步研究

采用RT-PCR技术从人的胎盘组织中克隆canstatin基因,定向连接到表达载体pUΩ上,然后与筛选标记bar盒连接得到真核表达载体pU Ω-Can-Bar。采用玻璃珠转化法将该表达载体转化杜氏盐藻(以下简称盐藻),通过草丁膦固体平板筛选得到转化株,进而对转化株进行阳性鉴定。PCR结果显示,在盐藻转化株中均能够扩增出约700 bp特异的条带,而在阴性对照中没有扩增出该条带。Southern blot结果进一步证明人canstatin基因已经整合到盐藻细胞的基因组中。此外,对盐藻转化株的遗传稳定性进行了分析,结果表明canstatin基因能够在转化藻株中稳定遗传。人canstatin转基因盐藻株的成功制备为利用盐藻反应器大规模生产人canstatin蛋白提供了实验依据,为及早实现canstatin蛋白在治疗肿瘤上的临床应用提供了前期工作基础。

Characterization and Expression Analysis of Protein Kinase C Gene from Dunaliella salina

Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.

根据β—胡萝卜素合成机制改进盐生杜氏藻(Dunaliella salina)培养方法的研究

根据β-胡萝卜素合成机制,在盐生杜氏藻(Dunaliella Salina)的培养过程中适当加入柠檬酸,Mg^(2+)和间断通CO_2可以提高盐生杜氏藻体内β—胡萝卜素的含量,其含量可达10.2％.

Effects of elevated pCO2 on physiological performance of marine microalgae Dunaliella salina (Chlorophyta, Chlorophyceae)

The present study was conducted to determine the ef fects of elevated pCO_2 on growth, photosynthesis, dark respiration and inorganic carbon acquisition in the marine microalga D unaliella salina. To accomplish this, D. salina was incubated in semi-continuous cultures under present-day CO_2 levels(390 μatm, p HN BS : 8.10), predicted year 2100 CO_2 levels(1 000 μatm, p HN BS : 7.78) and predicted year 2300 CO_2 levels(2 000 μatm, p H NBS : 7.49). Elevated pCO_2 significantly enhanced photosynthesis(in terms of gross photosynthetic O_2 evolution, ef fective quantum yield(ΔF/F' m), photosynthetic efficiency( α), maximum relative electron transport rate(r ETRm ax) and ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) activity) and dark respiration of D. salina, but had insignificant effects on growth. The photosynthetic O_2 evolution of D. salina was significantly inhibited by the inhibitors acetazolamide(AZ), ethoxyzolamide(EZ) and 4,4'-diisothiocyanostilbene-2,2′-disulfonate(DIDS), indicating that D. salina is capable of acquiring HCO ˉ 3 via extracellular carbonic anhydrase and anion-exchange proteins. Furthermore, the lower inhibition of the photosynthetic O2 evolution at high pCO_2 levels by AZ, EZ and DIDS and the decreased carbonic anhydrase showed that carbon concentrating mechanisms were down-regulated at high pCO_2. In conclusion, our results show that photosynthesis, dark respiration and CCMs will be af fected by the increased pCO_2/low p H conditions predicted for the future, but that the responses of D. salina to high pCO_2/low p H might be modulated by other environmental factors such as light, nutrients and temperature. Therefore, further studies are needed to determine the interactive eff ects of pCO_2, temperature, light and nutrients on marine microalgae.

Protective and therapeutic potentials of Dunaliella salina on aging-associated cardiac dysfunction in rats

Objective: To investigate the possible protective and/or therapeutic potentials of Dunaliella salina(D. salina) biomass, its carotenoid and polar fractions on cardiac dysfunction associated with D-galactose(D-GAL) induced aging in rats. Methods: Aging associated cardiac dysfunction was induced in rats by injection of D-GAL(200 mg/kg; i.p) for 8 weeks. D-GAL injected rats were treated with two regimens; protective regimen where D. salina biomass(250 mg/kg), its carotenoid(250 μg/kg) and polar(250 μg/kg) fractions were given orally for two weeks concurrently with D-GAL injection as well as treatment regimen where the three treatments were given orally for 28 consecutive days after D-GAL injection. Results: D-GAL injection for 8 weeks was accompanied with dramatic electrocardiographic changes as well as profound elevation in serum levels of homocysteine, creatinine kinase isoenzyme and lactate dehydrogenase in addition to the reduction of the cardiac content of glucose trasporter 4. D-GAL also induced reduction in cardiac superoxide dismutase activity and elevation of inducible nitric oxide synthetase and interleukin-6. On the other hand, oral administration of D. salina carotenoid fraction as well as the total biomass significantly attenuated the D-GAL-induced disturbances in the above mentioned parameters where the protective regimen appeared more successful in controlling the manifestations of cardiac dysfunction. The histopathological examination further emphasized the promising results. Besides, the HPLC analysis of the carotenoid fraction of D. salina revealed the presence of 2.31%. salina carotenoid fraction as well as the total biomass amelior β-carotene. Conclusions: Date D-GAL-induced aging associated cardiac dysfunction which is attributed to the potent antioxidant activity of β-carotene.

The effects of enriched C0_(2) and enhanced UV-B radiation on ultrastructure of Dunaliella salina, singly and in combination

The effects of ambient CO2/ambient UV-B, enriched CO2/ambient UV-B, ambient CO2/enhanced UV-B, and enriched CO2/enhanced UV-B on the ultrastructure of Dunaliella salina were investigated. （1） The ultrastructure of D..salina cell in the control experiment showed that the arrangement of thylakoid lamellae was regular, and there were many large starch grains among the thylakoid lamellae. A prominent well-developed pyrenoid was found in the middle of the chloroplast. Nucleus envelope and nucleolus were clearly observed. The Golgi apparatus accompanied by numerous vesicles with a compact arrangement of cisternae and the peripheral tips of the cisternae were swollen to a size comparable to that of some of the associated vesicles. （2） The ultrastructure of D. salina cell in enriched CO2 showed that the arrangement of thylakoid was regular and the lamellae were vivid. Developed pyrenoids were found in the low-CO2-grown cells, but not in the high-CO2-grown cells. The mitochondria cristae were vivid. The arrangement of Golgi apparatus was compact. （3） The ultrastructure of D. salina cell in enhanced UV-B showed that the thylakoid was dissolved and the cells had a less developed pyrenoid or no detectable pyrenoid. Part of the nucleus envelope was dissolved. The number ofmitochondria was increased and some mitochondria cristae were disintegrated. The starch grains were broken apart into many small starch grains. The Golgi apparatus with a loose arrangement ofcistemae and the peripheral tips of the Golgi cistemae were not especially swollen, with several large associated vesicles. （4） The ultrastructure of D. salina cell in the enriched CO2/enhanced UV-B showed that part of the thylakoid and nucleus envelopes of some cells were dissolved. The pyrenoid was larger than that of the enhanced UV-B. There were many mitochondria between stroma and chloroplast membrane, but mitochondria cristae were partly dissolved. Many small starch grains were accumulated in cells. The starch sheath was broken into several discontinuous starch grains with different sizes. The arrangement of Golgi apparatus was loose. Above all, although the enriched CO2 can alleviate the damage induced by the UV-B radiation, the effects of experimental UV-B radiation were larger than the effects of actual UV-B radiation, the damage induced by the UV-B radiation was so severe, therefore, CO2 enrichment could not restore the ultrastructure to the control level.

Transcriptome profile of Dunaliella salina in Yuncheng Salt Lake reveals salt-stress-related genes under diff erent salinity stresses

Salt stress is an abiotic stress to plants in especially saline lakes.Dunaliella,a halophilic microalga distributed throughout salt lakes and seas,can respond to different salinity stresses by regulating the expression of some genes.However,these genes and their function and biological processes involved remain unclear.Profi ling these salt-stress-related genes in a high-salt-tolerant Dunaliella species will help clarify the salt tolerance machinery of Dunaliella.Three D.salina_YC salt-stress groups were tested under low(0.51 mol/L),moderate(1.03 mol/L),and high(3.42 mol/L)NaCl concentrations and one control group under very low(0.05 mol/L)NaCl concentration and 3 transcriptome results that were deep sequenced and de novo assembled were obtained per group.Twelve high-quality RNA-seq libraries with 46585 upregulated and 47805 downregulated unigenes were found.Relative to the control,188 common differentially expressed genes(DEGs)were screened and divided into four clusters in expression pattern.Fifteen of them annotated in the significant enriched Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were validated via qPCR.Their qPCR-based relative expression patterns were similar to their RNA-seq-based patterns.Two significant DEGs,the geranylgeranyl diphosphate synthase coding gene(1876-bp cDNA)and diacylglycerol O-acyltransferase coding gene(2968-bp cDNA),were cloned and analyzed in silico.The total lipid content,superoxide dismutase specific activity,and betacarotene content of D.salina_YC increased gradually with increasing salinity.In addition,the expression of 11 validated genes involved in fatty acid biosynthesis/degradation,active oxygen or carotenoid metabolisms showed significant changes.In addition,algal photochemical efficiency was diminished with increasing salinity,as well as the expression of 4 photosynthesis-related genes.These results could help clarify the molecular mechanisms underlying D.salina responses to the Yuncheng Salt Lake environment and lay a foundation for further utilization of this algal resource.

Growth Enhancement of Dunaliella salina by Microbubble Induced Airlift Loop Bioreactor (ALB)—The Relation between Mass Transfer and Growth Rate

The efficiency of a novel microalgal culture system (an airlift loop bioreactor [ALB] engaged with a fluidic oscillator to produce microbubbles) is compared with both a conventional ALB (producing fine bubbles without the fluidic oscillator) and non-aerated flask culture. The impact of CO2 mass transfer on Dunaliella salina growth is assessed, through varying the gas (5% CO2, 95% N2) dosing flow rate. The results showed that approximately 6 - 8 times higher chlorophyll content was achieved in the aerated ALB cultures than in the non-aerated flasks, and there was a 20% - 40% increase in specific growth rate of D. salina in the novel ALB with microbubbles when compared with the conventional ALB cultures. The increase in chlorophyll content was found to be proportional to the total amount of CO2 mass transfer. For the same dosing time and flow rate, higher CO2 mass transfer rate (microbubble dosing) resulted in a greater growth rate.