Induction and characterization of green pigmentation mutant in Porphyra yezoensis Ueda

The free living conchocelis of Porphyra yezoensis Ueda was treated with N-methyl-N-nitro-N-nitrosoguanidine to induce pigmentation mutants. The artificial green pigmentation mutant of P. yezoensis conchocelis, which was composed entirely of green cells, was isolated through visualization with the unaided eye. The acquired green conchocelis was further developed into a green gametophytic blade. This mutant was relatively stable in color in both gametophytic blade and conchocelis phases. The gametophytic blade mutant was successively cultivated for commerce at some Porphyra farms in Rudong, China, and few wild type or sectorially variegated gametophytic blade occurred, indicating that the green mutant has commercial value. The green mutant was characterized as having lower phycoerythrin and higher phycocyanin content, and SDS-PAGE suggested that phycoerythrin was missing the gamma-subunit in comparison to the wild type. The wild type and the green mutant showed a clear difference in 02 evolution rates in white, green, yellow, and red light, which might be due to the qualitative and quantitative changes of phycoerythrin, and the quantitative difference of phycocyanin. (C) 2008 Elsevier B.V. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Comparative molecular population genetics of phycoerythrin locus in Prochlorococcus

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Expression of beta-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding beta-carotene hydroxylase that was able to catalyze the conversion of beta-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RTPCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.

Preparation of porous chitosan/agarose microsphere and its R-phycoerythrin release properties

The chitosan microspheres (CS-CL) were prepared by suspension crosslinking method and used as carriers of R-phycoerythrin (R-PE). In this study, R-PE was loaded in the microspheres and released in vitro. The effects of pH value, temperature, ionic strength, and R-PE concentration on loading efficiency and release behavior were discussed. A novel microsphere that contained agarose (CS-AR MP) was prepared and the basic loading and releasing behavior for R-PE of this kind of new micro-spheres were also investigated. The results showed that all these chitosan microspheres have the ability to control-release R-PE. The addition of agarose may somewhat accelerate the release rate of R-PE from microspheres and reduce the capacity of adsorption for R-PE. (c) 2006 Wiley Periodicals, Inc.

25ka B.P.以来黑潮流域古环境演化对高频全球变化事件的响应

本文利用黑潮流域主流轴上的两个柱状沉积物岩芯MD05-2908以及PC-1为研究材料，在AMS14C测年的基础上，利用高分辨率的有机地球化学分析记录结合浮游有孔虫氧、碳同位素，恢复并重建了过去25,000 cal a BP以来黑潮流域古海洋环境演化的历史。以及表层海水生产力以及物质输送状况的演化历史。通过利用Uk’37古海水温度以及盐度指标恢复重建了过去25,000 cal a BP以来海洋表层海水温度、盐度；通过机碳、氮含量以及同位素变化、长链正构烷烃以及正构烷醇等指标重建了过去7000a B.P.以来的陆源物质输入历史；通过长链不饱和烯酮含量以及有机碳同位素指标恢复了过去7000a B.P.以来海水表层生产力的历史。此外，通过基于以上各种指标的环境信息与区域以及全球其它气候记录进行对比研究以及不同环境指标的时间系列分析，探讨了该区表层环流系统以及生产力的演化对于全球气候变化的响应，揭示了不同尺度的短周期高频率全球变化事件在黑潮流域的具体作用过程和响应机制。通过这些研究取得了以下的主要认识： 基于有机地球化学指标的古气候环境记录与黑潮流域已有的研究成果有很好的对应关系，从我们高分辨率的有机地球化学记录中可以识别出全新世黑潮强弱变化的几次明显的事件；25000a B.P.以来黑潮流域的环境变化与全球环境变化有着很好的对应性，黑潮强弱演化总体趋势与全球气候背景演化相一致，黑潮对高频气候变化事件的记录与全球记录具有同步性，这种同步性尤其体现在末次冰消期以来的气候快速高频振荡以及全新世以来的气候突变事件上。 全球性的高频气候事件对黑潮主体本身及黑潮流域的相邻区域的大气和海洋环流都具有重要的控制作用，这种控制作用主要通过副高、ITCZ以及季风三种气候要素之间相互关联、彼此影响造成的。具体表现为：太阳辐射量的减少导致热力差异减小，这种相对减小弱化了热带西太平洋的对流活动，造成了西太平洋副热带高压长期偏南、偏东，ITCZ平均北界位置偏南，降雨带长时间集中在南部地区，增强的降雨量提高了风化剥蚀以及沉积物向海洋搬运的能力，陆源物质供应量增加；同时，辐射量以及热力差的减小又与加强的东亚冬季风相联系，增强的冬季风导致了近底层“雾状层”物质的向海传输，物质传输效率增高。这种物源供应以及搬运量的双重增加导致了冲绳海槽流域物质通量的增加。 基于有机地球化学指标的海洋表层生产力的变化与陆源物质供应量以及黑潮流的强弱变化存在着对应关系，通常情况增加的海洋表层生产力对应着高的陆源物质输入以及相对较弱的黑潮。这种变化与东亚夏季风的以及冬季风的强弱都有很好的一致性。陆源物质的输入增加了表层营养物质的含量，导致生产力的勃发；陆源物质的输入增加又对应着减少的太阳辐射量，偏南的ITCZ北界位置以及副热带高压，这些对应于减弱的东亚夏季风（增强的东亚冬季风）。 黑潮流域的高沉积速率事件对应于减弱的黑潮强度和增加的ENSO频度，这些事件与上述的副热带高压、ITCZ位移和强弱的变化相一致。黑潮流域过去25000a B.P.以来南北温度的差异有冰期加大而全新世减小的趋势，但这种冰期与全新世的差异很小，我们认为末次盛冰期的时候黑潮主流轴没有移出冲绳黑潮，只是由于强度的减弱受陆架水体的影响有所加大。 黑潮流域很好的记录到了包括数千年尺度的D/O旋回周期到大气——海洋系统内部振荡所致的PDO、NAO等数十年尺度的高频振荡，说明黑潮流域对过去全球及区域环境变化事件有很好的响应。黑潮流域各古海洋指标所记录周期上的一致性说明这些环境因子控制机理上具有的一致性，即大背景上受太阳活动所引起的辐射量变化控制，局部的高频快速气候波动又受到局域性的气候因素如海气相互作用的放大影响。

中国明对虾核转录因子NF-κB家族基因的克隆与表达分析

对虾流行病爆发以来，我国乃至世界的对虾产业一直受到各种虾病的困扰，对虾养殖业严重受阻。解决这一问题的关键是加强对虾免疫机制的研究，并在此基础上寻找对虾疾病防治的有效方法。Rel/NF-κB是一类核转录因子，在无脊椎动物的先天性免疫中，起着极为重要的作用。 本论文根据其他无脊椎动物中NF-κB家族基因Relish和Dorsal的保守氨基酸序列分别设计简并引物，从中国明对虾血细胞cDNA中先后克隆到了Relish和Dorsal基因的部分片段，并结合SMART-RACE技术分别获得了中国明对虾Relish基因（FcRelish）和Dorsal基因（FcDorsal）的cDNA 全长。 FcRelish的cDNA 全长为2157个碱基，其中开放阅读框为1512个碱基，编码504个氨基酸；FcRelish蛋白的推导分子量为57373.5 Da，理论等电点为7.00。FcDorsal基因的cDNA 全长为1627个碱基，其中开放阅读框为1071个碱基，编码357个氨基酸；FcDorsal蛋白的推导分子量为39780.7 Da，理论等电点为8.85。 分析了FcRelish基因和FcDorsal基因的在血细胞、淋巴器官、肠和肌肉等12个组织中的表达水平。组织表达结果表明FcRelish和FcDorsal在淋巴器官和血细胞中表达水平明显高于其他组织，而淋巴器官和血细胞是对虾免疫系统中最重要的两个组织，由此可以推测中国明对虾中的Relish和Dorsal可能与免疫关系密切。 本论文还利用实时荧光定量PCR技术，对灭活鳗弧菌刺激，以及WSSV病毒刺激后，对虾血细胞和淋巴器官中FcRelish基因和FcDorsal基因的转录水平进行了研究。FcRelish基因在WSSV病毒刺激后，在血细胞和淋巴器官中都出现了波浪形变化，说明FcRelish对WSSV病毒刺激产生了应答。在灭活鳗弧菌刺激后，FcRelish在血细胞中变化不明显，而在淋巴器官中出现了两次明显的下调上调交替，出现这种现象的具体原因有待探究。在WSSV病毒刺激后，血细胞和淋巴器官中FcDorsal的转录表达呈波浪形变化。而在鳗弧菌刺激后，FcDorsal在血细胞和淋巴器官中的转录均在短时间内出现明显的上调表达，说明FcDorsal对鳗弧菌非常敏感。 作为核转录因子的NF-κB蛋白的转录激活作用需要在细胞质中通过蛋白水解作用来激活，为了进一步阐明NF-κB对病菌感染的应答机制，需要进一步研究这两种转录因子在蛋白水平的变化，以便从分子水平阐明NF-κB在对虾天然免疫中的作用。

Proteasome and NF-kappa B inhibiting phaeophytins from the green alga Cladophora fascicularis

Chemical examination of the green alga Cladophora fascicularis resulted in the isolation and characterization of a new porphyrin derivative, porphyrinolactone (1), along with five known phaeophytins 2-6 and fourteen sterols and cycloartanes. The structure of 1 was determined on the basis of spectroscopic analyses and by comparison of its NMR data with those of known phaeophytins. Compounds 1-6 displayed moderate inhibition of tumor necrosis factor alpha (TNF-alpha) induced nuclear factor-kappa B (NF-kappa B) activation, while 2 and 4 displayed potential inhibitory activity toward proteasome chymotripsin-like activation. The primary structure-activity relationship was also discussed.

高寒草甸植物抗氧化系统对长期增强UV-B辐射的响应

通过研究自然条件下模拟平流层臭氧破坏5%时近地表面增加的太阳UV-B辐射对高寒草甸4种典型植物（矮嵩草Kobresia humilis、垂穗披碱草Elymus nutans、麻花艽Gentiana straminea和鹅绒委陵菜Po-tentilla anserina）的抗氧化系统的影响表明,尽管各植物的抗氧化系统组分变化不同,但4种植物的膜脂过氧化程度没有加剧,长期增强UV-B辐射没有对膜系统造成损伤。在自然长期增强UV-B条件下,4种植物的膜脂过氧化产物——丙二醛（MDA）含量与对照相比无显著差异。垂穗披碱草、鹅绒委陵菜的谷胱甘肽（GSH）含量增加,麻花艽的超氧化物歧化酶（SOD）活性与鹅绒委陵菜的过氧化氢酶（CAT）活性上升,同时麻花艽的类胡萝卜素（Car）含量亦显著增加。可见这些植物已能很好地适应UV-B强辐射,其抗氧化能力除了与抗氧化系统各组分的协同作用有关外,也可能与种的适应性有关。

祁连山海北高寒草甸地区UV-B的气候变化特征

分析祁连山海北高寒草甸地区2002年太阳总辐射（Eg）、UV-B及UV-B占Eg比例的气候变化特征。结果表明：海北站地区UV-B较强，日瞬时最高接近10W•m^-2，日总量最高达0．204MJ•m^-2；日、年变化依Eg的日、年变化具有显著的正相关关系。UV-B与Eg的比值（η），不论是日变化还是年变化表现明显，一日间早晚低，中午高，一年间6月最高，冬季的12月低，与太阳高度角的变化具有一定的正相关关系。年平均η约为0．54％，植物生长期的5～9月约为0．65％。在海北高寒草甸地区Eg和UV-B的年总量分别达6387．436MJ•m^-2和35．981MJ•m^-2。

长期增强UV-B辐射对高寒草甸植物光合速率和抗氧化系统的影响

研究了在野外自然条件下，长期增强UV-B辐射对高寒草甸3种典型植物矮嵩草（Kobresia humilis）、垂穗披碱草（Elymus nutans）和钉柱委陵菜（Potentilla saundersiana）光合放氧速率、光合色素和抗氧化系统的影响。结果表明：长期增强UV-B辐射对3种植物的净光合速率没有明显影响。增强UV-B辐射下，3种植物的叶绿素含量变化不同，Chla／b值，类胡萝卜素（Car）含量，Car／Chl值与对照相比都有升高，说明植物叶片的光合能力、吸收紫外线的能力增强以及忍受逆境能力均有增强，从而产生光保护，有利于光合作用正常进行。由于3种植物膜脂过氧化程度的不同及SOD活性的普遍抑制，植物经受了氧化胁迫。垂穗披碱草叶片GSH含量显著七升，矮嵩草的形态矮小及钉柱委陵菜GSH含量与POD活性显著上升，都能减轻它们所经受的氧化胁迫，使光合器官免受损伤。所以，这些保护性色素的积累和抗氧化系统内部的协同作用可能是高寒草甸植物光合作用正常进行的重要原因。

增强UV-B辐射对麻花艽叶片的抗氧化酶的影响

以青藏高原的特有植物麻花艽为材料,研究麻花艽叶片抗氧化酶系统对增强UV-B辐射的响应.结果表明:在UV-B处理初期,麻花艽叶片SOD、POD的酶活性都能增加,但随着处理时间的延长,SOD、POD的活性呈现下降趋势.麻花艽叶片CAT的酶活性在UV-B处理后下降明显,但作为清除叶绿体中H2O2的关键酶AP的酶活性表现为明显地增加趋势,说明在对麻花艽叶片增强UV-B辐射反应中AP起有着重要作用.MDA的含量随UV-B处理时间延长而增加,表明UV-B降低了细胞内活性氧自由基的清除能力,膜脂过氧化作用加剧,导致了对麻花艽叶片的伤害效应.

不同强度的UV-B辐射对高山植物麻花艽光合作用及暗呼吸的影响

以人工种植的多年生高山植物麻花艽( Gentiana straminea )为材料,在3个不同强度的UV-B辐射处理下,定时测定处理和对照叶片的净光合速率、表观量子效率和暗呼吸的变化.结果显示:UV-B处理对麻花艽叶片的光合作用在短期内有一定的抑制作用,但随着处理时间的增加,该高山植物能很快地适应强UV-B辐射的处理.表明麻花艽这种青藏高原常见的高山植物在长期的自然选择过程中可能已经形成了适应UV-B辐射的特有生理机制.暗呼吸的实验结果亦表明:在3种强度的UV-B辐射处理下,麻花艽叶片的呼吸作用从一开始就未受到抑制;随着UV-B辐射时间的增加,UV-B辐射强度越高,呼吸强度越强;这可能是 UV-B辐射并未引起麻花艽呼吸机构的破坏所致.