Mitochondrial NDP kinase from Dunaliella tertiolecta (Chlorophyceae, Chlorophyta)

A cDNA (DtNDK1) encoding a nucleoside diphosphate (NDP) kinase with a putative mitochondrial targeting signal sequence was previously isolated from the halo‐tolerant green alga Dunaliella tertiolecta. When expressed in Saccharomyces cerevisiae, the processed DtNDK1 enzyme was specifically localized to mitochondria. The present study reports several biochemical characteristics of the mitochondrial NDP kinase from D. tertiolecta. The mature protein was expressed as either N‐ or C‐terminal hexahistidine‐tagged protein and purified to homogeneity by affinity chromato‐graphy. Native gel electrophoresis and sedimentation velocity in sucrose density gradients showed that the active enzyme consisted of a hexamer. The enzyme, with a pH optimum of 7, showed the highest specificity to dCDP (K_m= 50 μmol/L) and the highest turnover towards the synthesis of UTP (up to 140‐fold higher). The present study also provides evidence that purified DtNDK1 proteins are capable of transferring a phosphate group to another protein.     

新型生物反应器——杜氏盐藻研究进展

转基因植物作为生物反应器生产外源物质已成为基因工程领域的研究热点之一 ,而杜氏盐藻 (Dunaliellasalina)作为新型生物反应器生产外源蛋白具有独特的优点 ,就盐藻这一生物反应器的特点、存在问题和开发应用前景等的最近研究进展作一简要综述。     

Oleic acid is the main fatty acid related with carotenogenesis in Dunaliella salina

The variation of the fatty acid profile and the carotene content of Dunaliella salina in response to irradiance (80, 128, 640, 1000, 1500 μmol photon m⁻² s⁻¹) and nitrogen starvation were analysed. The highest fatty acid content per cell and the least polyunsaturated fatty acid percentage were exhibited under 1500 μmol photon m⁻² s⁻¹. Furthermore, the oleic acid (18:1) content maintained a positive and significant correlation with the carotene content per cell and with the irradiance. The composition of the carotene globules in Dunaliella salina may be the main determinant of this correlation. This revised version was published online in June 2006 with corrections to the Cover Date.     

盐藻基因组DNA文库的构建(英文)

以ＬａｍｂｄａＦＩＸ○ＲⅡ为载体,构建了盐藻（Ｄｕｎａｌｉｅｌｌａｓａｌｉｎａ）的基因组文库。该文库包含了１．１×１０６个重组子,插入片段平均大小为１８ｋｂ左右,含插入片段的频率为１００％。该文库的容量约为盐藻单倍体基因组的２００倍。     

盐生杜氏藻对盐度改变的生理响应

以盐生杜氏藻为实验材料,采用f/2培养基,设置了8个盐度(15、20、25、30、50、70、90、110)处理,分盐度改变前(A)和盐度改变后(B)两个实验阶段,研究了盐生杜氏藻在不同盐度处理下的生长情况,测定了藻液的OD值、叶绿素a、β-胡萝卜素、可溶性蛋白质和可溶性糖含量等指标。结果表明,A阶段,几个较低盐度(15、20、25和30)处理生长状况较好,其中又以盐度20的处理最好;余下的处理,盐度越高,其生长所受的影响越大。B阶段,盐生杜氏藻的生长进入平台期后,50、70、90、110几个盐度较高处理的细胞密度、叶绿素a、β-胡萝卜素含量均显著超过了作为对照的盐度20的处理。且B阶段末期,先前盐度15的处理蛋白质、糖的积累量,与A阶段末期相比都有了不同程度的增加,而其余盐度处理组的蛋白质、糖含量则分别产生了不同程度的下降。     

Dunaliella salina (Chlorophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells

The photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta) cultures acclimated to different light intensities were investigated. Batch cultures were grown to the mid-exponential phase under continuous low-light (LL: 100 μmol photon m^-2 s^-1) or high-light (HL: 2000 μmol photon m^-2 s^-1) conditions. Under LL, cells were normally pigmented (deep green) containing ∼500 chlorophyll (Chl) molecules per photosystem II (PSII) unit and ∼250 Chl molecules per photosystem I (PSI). HL-grown cells were yellow-green, contained only 60 Chl per PSII and 100 Chl per PSI and showed signs of chronic photoinhibition, i.e., accumulation of photodamaged PSII reaction centers in the chloroplast thylakoids. In LL-grown cells, photosynthesis saturated at ∼200 μmol photon m^-2 s^-1 with a rate (P_max) of ∼100 mmol O₂ (mol Chl)^-1 s^-1. In HL-grown cells, photosynthesis saturated at much higher light intensities, i.e. ∼2500 μmol photon m^-2 s^-1, and exhibited a three-fold higher P_max (∼300 mmol O₂ (mol Chl)^-1 s^-1) than the normally pigmented LL-grown cells. Recovery of the HL-grown cells from photoinhibition, occurring prior to a light-harvesting Chl antenna size increase, enhanced P_max to ∼675 mmol O₂ (mol Chl)^-1 s^-1. Extrapolation of these results to outdoor mass culture conditions suggested that algal strains with small Chl antenna size could exhibit 2–3 times higher productivities than currently achieved with normally pigmented cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

不同启动子驱动下转基因盐藻外源基因的稳定表达

摘要：为了探讨外源性与内源性启动子对转基因盐藻外源基因表达的影响,将含外源性启动子CMV35S的表达载体CMV35S-bar（G12）和含内源双拷贝碳酸酐酶启动子DCA1的表达载体DCA1-bar（D-B）分别转化盐藻,筛选稳定转化株后,观察在不同启动子驱动下外源基因的表达情况及对转基因盐藻生长的影响。 通过电击法分别将表达载体G12 和D-B转化盐藻,经PPT筛选后,各得到了3株PPT抗性藻株,经PCR及测序分析证实外源基因bar已经整合到盐藻的基因组中,半定量RT-PCR结果显示,在内源性启动子DCA1驱动下,bar基因的表达强度明显高于在外源性启动子驱动下bar的表达,并且D-B转化株的bar基因表达在盐诱导下其表达明显提高,而G12转化株中bar基因的表达对盐诱导无反应。Southern blot 分析显示,外源基因的拷贝数与不同启动子间无相关性。转化株的生长特性分析显示,D-B转化株的生长速度明显高于G12转化株。本研究的结果指出,内源诱导型启动子在驱动转基因盐藻外源基因的高效稳定表达中比外源组成型启动子更具有优势。     

绿藻Dunaliella salina钙依赖蛋白激酶的特征(英文)

通过凝胶过滤从绿藻Dunaliella salina中分离出一种新类型的钙依赖但非钙调素、磷脂依赖的蛋白激酶,用凝胶过滤法测得此酶的天然分子量为52kD。该酶的活性既依赖于Ca~(2 )也需要Mg~(2 ),最适浓度为4mmol/L。NaCl和KCl对酶活性具抑制作用。蛋白酶抑制剂K-252a和staurosporine可抑制该酶活性,但半抑制浓度 (IC_(50))远高于对蛋白激酶C(PKC)。当PKC专一性抑制剂sphingosine浓度高达800μmol/L时,对该酶只表现微弱的抑制效应。碱性的组蛋白H1为所测定的蛋白质底物中最适的底物,而酸性的酪蛋白不被磷酸化。磷酸化氨基酸残基分析表明,该酶属丝氨酸/苏氨酸型蛋白激酶。     

Phylogenetic analysis of ITS2 sequences suggests the taxonomic re‐structuring of Dunaliella viridis (Chlorophyceae,Dunaliellales)

We analyzed the ITS2 primary and secondary structure (including Compensatory Base Changes (CBCs)) of 17 new Dunaliella strains (11 D. viridis, two D. tertiolecta, and four Dunaliella sp.), and compared these with other Dunaliella sequences available from the ITS2 database to circumscribe their taxonomic position. The ITS2 primary and secondary structure analysis positioned the majority of D. viridis strains in four main clades, showing that D. viridis is polyphyletic. The detection of at least one CBC among these clades strongly suggests that they could correspond to different biological species. Unexpectedly, while D. viridis var. euchlora (CCAP19/21) was positioned within the subgenus Dunaliella, D. viridis var. palmelloides (CCAP11/34) was positioned clearly outside this subgenus, suggesting that this taxon may not be properly placed in Dunaliella. Furthermore, the detection of at least three compensatory base changes (CBCs) between D. viridis var. palmelloides (CCAP11/34) and the other strains analyzed, confirm that this strain is a different species. For these reasons we propose re‐naming D. viridis var. palmelloides (CCAP11/34) to incertae sedis, and D. viridis var. euchlora (CCAP19/21) to Dunaliella sp. Therefore, the ITS2 primary and secondary structure data suggest a taxonomic re‐structuring of D. viridis.     

杜氏盐藻渗透调节过程中的甘油代谢途径

杜氏盐藻在适应外界盐浓度变化的过程中,甘油是其主要的渗透调节物质。低渗处理提高藻细胞的呼吸速率６０％以上;高渗处理对呼吸无明显影响,但大大刺激光合放氧速率。呼吸链的细胞色素电子传递链抑制剂ＫＣＮ和交替氧化酶抑制剂ＳＨＡＭ对杜氏藻渗透调节过程中的呼吸．胞内甘油、ＡＴＰ、淀粉会量的变化有不同的抑制效果。低渗情况下,胞内甘油转化为淀粉,所需能量由正常呼吸链和交替氧化酶途径同时提供;高渗情况下．淀粉则降解为甘油,光下甘油合成的能量主要由光合电子链提供,暗中则由正常呼吸链提供。     

Stomatal responses to jasmonic acid, linolenic acid and abscisic acid in wild-type and ABA-deficient tomato plants

Wild-type and abscisic acid (ABA) -deficient (sitiens) tomato plants were used to analyse the effects of abscisic acid (ABA), butyric acid (BA), jasmonic acid (JA) and linolenic acid (LA) on assimilation and transpiration rates in detached leaves taking up those substances into the transpiration stream. BA did not affect assimilation and transpiration rates. ABA decreased assimilation and transpiration in both wild-type and ABA-deficient mutants. JA reduced the assimilation rate in both lines but induced a significant reduction of transpiration in the wild type only. The response to LA in both lines was slower than that to JA.     

Purification and characterization of an algal (Dunaliella tertiolecta) protein cross-reacting with an anti-Ga-common antibody

A 26-kDa and a 36-kDa protein that cross-reacted with anti-G_a-common and anti-G_β antibodies, respectively, were detected in Dunaliella cells. The 26-kDa protein was solubilized from a crude membrane fraction with deoxycholate and purified to homogeneity by DE52 and hydroxylapatite chromatography and DEAE-5PW high performance liquid chromatography (HPLC). The hydroxylapatite-purified preparation had GTPγS binding and GTPase activities, but the homogeneous 26-kDa protein had none. The sequence of the 28 N-terminal amino acids of the 26-kDa protein had no homology to any GTP binding protein thus far reported.     

水杨酸、茉莉酸和乙烯介导的防卫信号途径相互作用的研究进展

乙烯、水杨酸和茉莉酸是植物体内主要的几个防御信号途径,也是研究比较多的几个信号途径。很多试验证明不同的防御信号途径相互间存在相互作用,他们或相互抑制,或相互促进。从这三种信号途径相互间的作用,及作用的联系点进行综述。     

Circadian changes in endogenous concentrations of indole-3-acetic acid,melatonin, serotonin,abscisic acid and jasmonic acid in Characeae (Chara australis Brown)

Giant-celled Characeae (Chara australis Brown), grown for 4 months on 12/12 hr day/night cycle and summer/autumn temperatures, exhibited distinct concentration maxima in auxin (indole-3-acetic acid; IAA), melatonin and serotonin about 4 hr after subjective daybreak. These concentration peaks persisted after 3 day pretreatment in continuous darkness: confirming a circadian rhythm, rather than a response to “light on.” The plants pretreated for 3 d in continuous light exhibited several large IAA concentration maxima throughout the 24 hr. The melatonin and serotonin concentrations decreased and were less synchronized with IAA. Chara plants grown on 9/15 hr day/night cycle for 4 months and winter/spring temperatures contained much smaller concentrations of IAA, melatonin and serotonin. The IAA concentration maxima were observed in subjective dark phase. Serotonin concentration peaks were weakly correlated with those of IAA. Melatonin concentration was low and mostly independent of circadian cycle. The “dark” IAA concentration peaks persisted in plants treated for 3 d in the dark. The plants pretreated for 3 d in the light again developed more IAA concentration peaks. In this case the concentration maxima in melatonin and serotonin became more synchronous with those in IAA. The abscisic acid (ABA) and jasmonic acid (JA) concentrations were also measured in plants on winter regime. The ABA concentration did not exhibit circadian pattern, while JA concentration peaks were out of phase with those of IAA. The data are discussed in terms of crosstalk between metabolic pathways.     

杜氏盐藻的耐盐机制研究进展和基因工程研究的展望

概述了杜氏盐藻 (Dunaliellasalina)的耐盐机制和基因工程的研究进展。盐藻的耐盐机制十分复杂 ,短时间内通过细胞体积的改变来调节渗透压平衡 ,之后通过甘油的合成与转化恢复细胞正常形态和大小。渗透调节过程中 ,还涉及到蛋白质的合成。cDNA文库和基因组文库已经建立 ;几种基因已被克隆 ,如碳酸酐酶基因和硝酸还原酶基因等 ;GUS(β_葡糖苷酸酶 )基因已成功地转入盐藻细胞内。另外 ,对盐藻的基因工程作了简单的展望     

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

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CHROMA SPIN-400柱子将cDNA分段化,最后将长片段连入pDNR-LIB质粒,1.5 kV,25 μ F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1.5×106cfu ml-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.     

Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress

Abstract Dunaliella tertiolecta cells subjected to a hyperosmotic shock of 0.930 osmol kg^?1 start almost immediately to synthesize glycerol at a rate of some 100 nmol min^?1 mg protein^?1. Glycerol synthesis was equally fast in both light and darkness, and was not affected by the nature but only by the concentration of solutes. During the period of rapid glycerol synthesis, which lasted about 1h, the concentration of glycerol-3-phosphate transiently increased. During the same period, ATP, fructose 1-6-bisphosphate, and triosephosphate content decreased markedly, especially when 0.1 kmol m^?3 NaCl-grown cells were used. The content of hexose-6-phosphates, nicotinamide coenzymes, and phosphate underwent no dramatic changes. Since no in vitro activity changes of the glycerol cycle enzymes could be detected during the adaptation period, the activity of glycerol-3-phosphate dehydrogenase in vivo is probably increased by a change in concentration of its effectors such as ATP.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Cr3+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^-16个添加Cr³⁺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、â胡萝卜素和可溶性糖含量.研究结果表明,中低量添加Cr³⁺对盐藻的生长有一定的促进作用,在50ìg·L^-1和200ìg·L^-1Cr³⁺条件下,盐藻的生物量高于对照,中低量添加Cr³⁺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%,Cr³⁺浓度在200ìg·L^-1时,盐藻的â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Cr³⁺可提高盐藻蛋白质、â胡萝卜素和可溶性糖含量,有效改善盐藻的营养品质.