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.

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。

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。

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。

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％.

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。

K^+浓度对杜氏盐藻(Dunaliella salina)生长的影响

为探究杜氏盐藻生长所需的适宜K+浓度,本文在实验室条件下,研究了杜氏盐藻(Dunaliella salina)在不同K+浓度(1、4、8、12 mmol/L)下生长增殖变化特征。对各K+浓度下,杜氏盐藻第11天的细胞密度进行单因素方差分析和多重比较,结果表明,只有8mmol/L和12 mmol/L之间为显著性差异(0.01<P<0.05),其余各组间均为极显著差异(P<0.01)。杜氏盐藻生长所需的适宜K+浓度为2 mmol/L。

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.

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.

盐生杜氏藻Dunaliella salina的生物学特性与培养研究

盐生杜氏藻富含β－胡萝卜素和蛋白质，适宜在高纬度、光照强的各种咸水环境中生长．对它的生物学特性和培养条件进行了研究，发现适宜于盐生杜氏藻生长的培养基主要成份为：２ｍｏｌ／ＬＮａＣｌ，５ｍｍｏｌ／ＬＫＮＯ＿３，６．５ｍｍｏｌ／ＬＮａＨＣＯ＿３，５ｍｍｏｌ／ＬＭｇＳＯ＿４，０．３ｍｍｏｌ／ＬＫＨ＿２ＰＯ＿４．

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.

杜氏盐藻(Dunaliella salina)对重金属铜胁迫的生理响应

将不同质量浓度的CuCl_2溶液添加到培养基中,研究重金属铜对杜氏盐藻(Dunaliella salina)胁迫的生理响应.分别用不同质量浓度的CuCl_2溶液培养盐藻,检测盐藻的光合色素、可溶性多糖、蛋白质、SOD及MDA质量浓度的变化.结果显示,铜可抑制盐藻细胞的生长,且呈剂量-效应关系,72 h的EC50为9. 55 mg/L.随着铜质量浓度的增加,各质量浓度组盐藻细胞内叶绿素a、b、总叶绿素、类胡萝卜素、多糖和蛋白质质量浓度均显著性降低(P <0. 05或P <0. 01).铜可导致盐藻细胞氧化损伤,随着铜质量浓度的升高,SOD活力先升高后降低,MDA质量浓度升高.表明铜可以抑制盐藻细胞内光合色素、多糖和蛋白质的合成.盐藻对铜胁迫的响应机制可能是通过抗氧化防御系统.

CO2加富对盐生杜氏藻(Dunaliella salina)叶绿素荧光参数的影响

大气中CO_2浓度不断升高导致的海水酸化,已经引起了广泛的环境、生态和气候问题。本实验采用实验生态学的方法,以盐生杜氏藻(Dunaliella salina)为研究对象,分析其在CO_2加富的条件下叶绿素荧光参数的变化。研究表明,CO_2加富对盐生杜氏藻光系统Ⅱ最大光化学量子产额(Fv/Fm)和最大相对电子传递速率(rETRmax)无显著影响(P>0.05),显著促进了光系统Ⅱ实际光合效率(P<0.05)和光能利用效率(α)(P<0.05),并且降低了饱和光强(Ek)(P<0.05)。然而,CO_2升高增加了盐生杜氏藻的光抑制参数(β)(P<0.05)和非光化学淬灭(NPQ)(P<0.05),这说明在光照充足的情况下,CO_2加富会对盐生杜氏藻产生负面效应,使其更容易受到光抑制。

不同培养基对杜氏藻（DunaliellaSalina）生长和无机离子含量的影响

培养在Ｊｏｈｎｓｏｎ培养液、Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液、海水和卤水中的杜氏藻，其生长速度有区别，在Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液中生长较好，Ｊｏｈｎｓｏｎ培养液和卤水次之，海水中生长较差。杜氏藻生沃的盐度范围为０～１２％，当培养基中ＮａＣｌ浓度超过１２％时，细胞数几乎不增加，甚至略有降低。在不同培养基中藻细胞Ｈ￣＋含量较稳定，而积累ｃａ￣（２＋），在Ｊｏｈｎｓｏｎ＋０．３％ＮａＣｌ培养液中，杜氏藻细胞Ｎａ￣＋含量增加；而在含高浓度Ｎａ￣＋的海水和卤水中杜氏藻细胞中Ｎａ￣＋的含量低于培养液。

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.

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.

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.