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。

Establishment of a New Method for Genetic Transformation of Dunaliella salina

The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing that the exogenous GUS gene was successfully expressed in the cells of D. salina. Meanwhile, the effects of growth state of D. salina, plasmid concentration and temperature on its transformation efficiency were studied, and the transformation conditions were optimized. The results show that the optimum conditions for the genetic transformation of D. salina are shown as follows： D. salina was in the early logarithmic phase; plasmid DNA concentration was 600 μg/ml; temperature was 29 ℃, and transformation efficiency was up to 74.8‰ under the best conditions. According to the results of PCR amplification and PCR-Southern hybridization, the target gene had been integrated into genome and was hereditary.

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。

Heterocypris salina生物学研究

Heterocyprissalina是一类小型甲壳动物，其寿命一般为45d，从孵化长至成体需22（±2）d，在生长过程中外壳无变化，但是附肢有明显变化。同时有蜕壳现象，一般要经过8次蜕壳。产卵数为38（±3）个，孵化率为87％左右。日．salina为广盐广温动物，其温度耐受范围为8～44℃，生活温度范围为16～38℃；盐度耐受范围为0％一33‰，生活盐度范围为o％一19％o。

Cloning, Analysis and Prokaryotic Expression of DsSP Gene from Dunaliella salina

[Objective] The purpose of this study was to clone a starch phosphorylase gene from Dunaliella salina and to preliminarily analyze its basic properties and protein expression. [Method] RT-PCR and RACE （rapid amplification of cDNA ends） method was used for gene cloning; basic properties of the gene were analyzed using bioinformatics method; prokaryotic expression vector PGS21a-DsSP was constructed and transformed into E. coil BL21; the fusion protein was purified and detected by GST-SefinoseTM Kit and Western Blot, respectively. [Result] A starch phos-phorylase gene （GenBank accession No. KF061044） named DsSP was successfully isolated from D. salina. Basic properties, subcellular localization, secondary structure and tertiary structure of the protein were analyzed and predicted. The fusion protein was found in the supernatant and inclusion bodies. The supernatant protein was successfully purified. Western Blot analysis showed that the fusion protein was successfully expressed in E. coil BL21. [Conclusion] This study laid experimental foun- dation for further clarifying the function and mechanism of DsSP.

Advances in Studies on Molecular Mechanism of Salt Tolerance in Dunaliella salina

Dunaliella salina is known as one of the most salt-tolerant eukaryotic or- ganisms, and the most ideal model organism for studying plant adaption to high salinity. In recent years, the study on molecular mechanism of salt tolerance in Dunaliella salina has become the focus of scholars at home and abroad with the development of molecular biological techniques. This study reviewed studies on adaption of Dunaliella salina to high salinity in aspects of osmotic adjustment, salt tolerance-related genes and proteins of Dunaliella salina and signal transduction pathway of salt stress.

溶气气浮法采收盐藻(Dunalilla salina)细胞

初步探讨了不添加絮凝剂和表面活性剂时用溶气气浮法采收盐藻细胞的工艺条件,结果表明,藻液pH值、溶气压(PS)、溶气水进水流量(QW)、溶气水/原料藻液体积比(水液比αWF)对采收效果有重要影响. 本实验条件下,调节藻液pH值为11.5, 在PS＝0.39 MPa, QW＝7 ml/s, αWF＝1.5的气浮操作条件下,细胞采收率和浓缩倍数分别达86.4%～96.4%和2.38～5.57. 表明气浮法可以安全、高效地从培养液中采收盐藻细胞,无需添加絮凝剂.

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。

Prokaryotic Expression and Purification of Ds MAPK from Dunaliella salina

Dunaliella salina is an important model organism for investigating the salt tolerance mechanism of plant. MAPK is the key gene in the molecular pathway of salt tolerance of plant. In this experiment, the open reading frame （ORF） of DsMAPK gene was amplified by PCR. The target fragment was cloned in pGS-21a, a prokaryotic expression vector with GST-tag. The recombinant plasmid pGS-21a- DsMAPK was then transformed into E. coil BL21 （DE3）. The expression was induced with IPTG. Then the expression form of the recombinant protein was analyzed. The expression products were purified with GST-SefinoseTM Kit and identified with SDS-PAGE and Western blot. The results showed the recombinant expression vector pGS-21a-DsMAPK was constructed successfully, and the molecular weight of the expressed recombinant protein was as same as expected. Western blot analysis showed the purified recombinant protein could be identified specially by the anti- GST antibody and had a good immunological activity. The successful expression of DsMAPK will lay a basis for the further research on the role of DsMAPK in the salt tolerance mechanism at the protein level.

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。

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.

Interspecific competition and allelopathic interaction between Karenia mikimotoi and Dunaliella salina in laboratory culture

Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella salina in laboratory cultures based on different temperature （15℃, 20℃, and 25℃） and lighting （40,80, and 160 umol/（m2·s）） conditions. The growth of D. salina in bi-algae culture （1：1 size/density） was significantly restrained. The results of cell-free filtrate culture indicate that direct cell-to- cell contact was not necessary in interspecific competition. Further experimental results demonstrated that allelochemicals released from K. miMmotoi were markedly influenced by both temperature （P=0.013） and irradiance （P=0.003）, resulting in different growth characteristics olD. salina in filtrate mediums. Compared with the plateau period, K. mikimotoi exudates in the exponential phase had a stronger short-term inhibition effect on D. salina in normal conditions. A clear concentration-dependent relationship was observed in the effect of allelochemicals released from K. mikimotoi with low-promoting and high-repressing effects on D. Salina in a short time-scale. In addition, allelopathic substances remain stable and effective under high temperature and pressure stress. Many flocculent sediments adhering with D. salina cells were observed in all filtrate mediums, while the quantity and color depended on the original culture conditions.

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％.

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.

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

The present study was conducted to determine the effects of elevated pCO2 on growth, photosynthesis, dark respiration and inorganic carbon acquisition in the marine microalga Dunaliella salina. To accomplish this, D. salina was incubated in semi-continuous cultures under present-day CO2 levels （390 μatm, PHNBs： 8.10）, predicted year 2100 CO2 levels （1000 μatm, pHNBs： 7.78） and predicted year 2300 CO2 levels （2 000μatm, PHNBS： 7.49）. Elevated pCO2 significantly enhanced photosynthesis （in terms of gross photosynthetic O2 evolution, effective quantum yield （△F/F＇m）, photosynthetic efficiency （a）, maximum relative electron transport rate （rETRmax） and ribulose-1,5-bisphosphate carboxylase/ oxygenase （Rubiseo） activity） and dark respiration olD. salina, but had insignificant effects on growth. The photosynthetic 02 evolution olD. salina was significantly inhibited by the inhibitors acetazolamide （△Z）, ethoxyzolamide （EZ） and 4,4＇-diisothiocyanostilbene-2,2＇-disulfonate （DIDS）, indicating that D. salina is capable of acquiring HCO3 via extracellular carbonic anhydrase and anion-exchange proteins. Furthermore, the lower inhibition of the photosynthetic O2 evolution at high pCO2 levels by AZ, EZ and DIDS and the decreased carbonic anhydrase showed that carbon concentrating mechanisms were down-regulated at high pCO2. In conclusion, our results show that photosynthesis, dark respiration and CCMs will be affected by the increased pCO2/low pH conditions predicted for the future, but that the responses olD. salina to high pCO2/low pH might be modulated by other environmental factors such as light, nutrients and temperature. Therefore, further studies are needed to determine the interactive effects ofpCO2, 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.