盐胁迫下杜氏盐藻(Dunaliella salina)cDNA文库的构建与新表达序列标签(EST)的获取、分析

在成功养殖杜氏盐藻(Dunaliella salina)的基础上,采用Takara cDNA文库构建试剂盒,构建盐胁迫下(1.5 mol/L NaCl)杜氏盐藻的cDNA文库.经鉴定,原始文库的滴度达1.2×106cfu/mL,重组率高达95%,且多数插入片段均在500 bp以上.对表达序列标签(expressed sequence tag,EST)分析发现,杜氏盐藻基因组中包含大量未鉴定的新基因.随机挑取60个单克隆进行序列测定,将所测序列经Blast比对等生物信息学方法分析后,发现其中三分之一,即20

Cell responses of Dunaliella salina FACHB 435 (green alga) to microgravitational stimulation by clinorotation

Clinorotation experiments were established to simulate microgravity on ground. It was found that there were obvious changes of Dunaliella salina FACHB435 cells and their metabolic characteristics during clinorotation. The changes included the increases of glycerol content, the rate of H+ secretion and PM H+-ATPase activity, and the decrease of ratio of the plasma membrane (PM) phospholipid to PM protein. These results indicated that microgravity was a stress environment to Dunaliella salina. It is deduced that it would be possible to attribute the effect of microgravity on algal cells to the secondary activation of water stress.

Establishment of a micro-particle bombardment transformation system for Dunaliella saliva

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. saliva with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 mu g/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Algal biotechnology industries and research activities in China

In old China there were very few people engaged in the study of the algae, but in new China, freshwater and marine algae are studied by over one hundred old and new phycologists. There is now an algal biotechnology industry consisting of an aquaculture industry, producing large amounts of the seaweeds Laminaria, Porphyra, Undaria, Gracilaria, eucheumoids, and the microalgae Dunaliella and Spirulina. There is also a phycocolloid industry, producing algin, agar and carrageenan; an industry producing chemicals and drugs, such as iodine, mannitol, phycocyanin, beta -carotene, PSS (propylene glycol alginate sulfate) and FPS (fucose-containing sulfated polysaccharides) and an industry producing food, feed and fertilizer. The Laminaria cultivation industry produces about 900,000 t dry Laminaria, probably the largest producer in the world and 13,000 t algin, undoubtedly one of the largest algin producer in the world.

Effects of various algal diets and starvation on larval growth and survival of Meretrix meretrix

Effects of food availability on larval growth and survival of Meretrix meretrix were studied in two experiments by feeding the larvae with different algae diets and by starving the larvae for different periods of time. Newly hatched larvae of M meretrix were fed with five different marine microalgae species, singly and in various mixtures. Best growth was with Isochrysis galbana as a single species diet. Nutritional value of the other single species diets was in the order of Dunaliella sp.> Phaeodactylum tricornutum > Platymonas subcordiformis > Pavlova viridis. Of the mixtures tested, 50% I. galbana/50% Dunaliella sp., 50% I. galbana/50% P tricornutum, and 50% 1 galbana/50% P subcordiformis, supported growth and metamorphosis equivalent to those of the I. galbana control. At 25 degrees C, larvae of M meretrix were deprived of food for various days to study the growth compensation from the outset of development. The results showed that M meretrix larvae could survive long feeding delays, and even reach metamorphosis without food added, although starvation had significant effects on growth. These results suggested that M meretrix larvae had the capacity to survive 'starvation' using alternative sources of energy. It also showed that growth, survival and metamorphosis of M meretrix were affected by many factors besides food quality and quantity. (c) 2005 Elsevier B.V. All rights reserved.

胁迫要件下盐藻β－胡萝卜素及其异构体累积的研究

为弄清胁迫条件对盐藻β－胡萝卜素异构体累积的影响，寻找养殖盐藻生产β－胡萝卜素的适宜条件。我们以盐生杜氏藻（Dunaliella salina. Teodoresco，简称盐藻）为实验材料，利用海水，卤水和苦卤水为培养液，在室内人工控制条件或室外自然条件下，进行藻体培养，1－2周后，收集藻体，计算细胞数目，提取色素，萃取色素，旋转蒸干，柱层析提纯，分光比色和高效液相色谱分析。首次研究了盐度、氮、磷、温度对盐藻β－胡萝卜素异构体累积的影响，同时还研究了光对异构体累积的影响。并且系统地研究了盐度、温度、光强、氮、磷、铁、碳、气体、苦卤水等对细胞生长、β－胡萝卜素累积的影响。上述实验获得一系列实验结果，这些结果为大规模生产性养殖打下了理论基础。其中部分结果属国内外首次报道：1.盐藻至少可以积累六种β－胡萝卜素异构体。2.氮浓度降低有利于β－胡萝卜素顺式异构体累积，其中对总顺式异构体累积来说，1mM KNO_3较为适宜。3.磷浓度降低有利于总顺式异构体的累积，其中以0.1mM KH_2PO_4较为适宜。4.温度适当升高有利于总顺式异构体的累积。5.盐度增高不利于总顺式异构体的累积，而有利于全反式异构体的累积。6.盐藻累积β－胡萝卜素越多，藻体越能忍耐高温胁迫。大量累积β－胡萝卜素的藻体能耐48.5 ℃ 2小时而不死亡。恒温对细胞生长不利。7.藻液中通入空气不利于细胞生长，也不利于β－胡萝卜素的积累。8.也可以利用苦卤水养殖盐藻。卤水中加入苦卤水后细胞生长加快，但β－胡萝卜素在细胞内的累积量降低。若培养液中加苦卤水占18.5％或75％培养效果较为理想。在获得上述实验结果基础,上我们总结出各种胁迫因子对细胞生长、β－胡萝卜素及其顺式异构体累积影响的一览表。并设计出三种模式：1>有利于细胞生长的模式，2>有利于β－胡萝卜素累积的模式，3>有利于顺式异构体累积的模式。

环境因子对养殖生物白斑综合症传播和分子生物标志物系统的影响研究

本文研究了海洋微藻在白斑综合症(white spot syndrome)暴发中的可能作用，以及阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)长期暴露对紫贻贝(Mytilus galloprovincialis)生物标志物系统的影响(72 d)。 1．海洋微藻在养殖对虾白斑综合症传播中的作用研究 为了证实海洋微藻是否是养殖对虾白斑综合症的传播途径，我们首先将六种海洋微藻：球定边金藻(Isochrysis galbana)、中肋骨条藻(Skeletonema costatum)、小球藻(Chlorella sp. )、赤潮异湾藻(Heterosigma akashiwo)、锥状斯氏藻(Scrippsiella trochoidea)和盐藻(Dunaliella salina)，与人工注射感染白斑病毒(white spot syndrome virus)的成体日本囊对虾共同培养，用套氏PCR方法检测共培养的微藻能否携带白斑病毒。在此基础上，进一步研究了共培养后的海洋微藻是否能感染幼体日本囊对虾。研究结果表明，除了H. akashiwo，实验海洋微藻均可携带白斑病毒，但它们携带病毒的能力有明显差异，Chlorella sp.和S. trochoidea携带白斑病毒的能力较强；但是，与白斑病毒的其他携带者（如桡足类等）不同，携带病毒的海洋微藻10天后病毒检测结果均呈阴性。共培养后小球藻组可感染幼体日本囊对虾，但幼体携带病毒的量只能通过二步PCR方法才能检测到。上述结果表明，海洋微藻在WSSV的水平传播途径中具有一定的作用。 2．表面活性剂对紫贻贝生物标志物系统的影响研究 以青岛胶州湾现场调查数据为依据，选择阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)作为污染物、以近海底栖生物紫贻贝为受试生物，研究了长期暴露后紫贻贝生化指标(SOD, CAT, GSH, GPx, GST, iNOS, AKP)和遗传毒理指标(AFLP指纹图谱)的变化。实验结果发现: 经过72d不同浓度暴露后，SDBS实验组紫贻贝体内的SOD、CAT和iNOS活性均有显著下降（除CAT 0.1mg/L组外），GSH、GST和GPx在3.0mg /L SDS、SDBS组较各自对照组均有显著升高。SDBS对紫贻贝生化指标影响的显著性水平大于SDS。统计分析显示，SDBS暴露组下GST与GPx呈显著正相关关系，iNOS与SOD也表现出一定正相关,但GSH与CAT、GSH与SOD呈现显著负相关关系。SDS浓度与GST呈显著正相关，而SDBS浓度与CAT呈显著负相关。另外，实验结果发现后闭壳肌中iNOS是一个具有应用前景的阴离子表面活性剂暴露生物标志物。AFLP标记结果统计显示，在实验给定的污染物浓度下，SDBS基因毒性要大于SDS；不同的DNA指纹图谱以及遗传距离图显示不同的污染物造成的DNA损伤是不同的。结果表明，在长期暴露条件(72 d)下，一定浓度的阴离子表面活性剂可以对岗哨生物紫贻贝的SOD, CAT, GSH, GPx, GST, iNOS和AFLP指纹图谱一组指标产生显著影响。

Studies on the transmission of WSSV (white spot syndrome virus) in juvenile Marsupenaeus japonicus via marine microalgae

We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microallgae (Isochr.vsis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japollicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after I and 3 days, were negative after 10 days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae. (c) 2007 Elsevier Inc. All rights reserved.

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293 parts per thousand) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 x 13 mu m in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).