Characterization of the main light-harvesting chlorophyll a/b-protein complex of green alga, Bryopsis corticulans

The main light-harvesting chlorophyll a/b -protein complex (LHC II) has been isolated directly from thylakoid membranes of shiphonous green alga, Bryopsis corticulans Setch. by using two consecutive runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC 11 were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin, violaxanthin and siphonaxanthin were involved in LHC 11 from B. corticulans. The properties of electronic transition of monomeric LHC II showed similarities to those of trimeric LHC II. Circular dichroism spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and between Chl b exist in one LHC II apoprotein, while the intermolecular interaction of these dipoles can be intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHC II monomer has high ordered pigment organization that play effective physiological function as the trimer, and thus it might be also a functional organization existing in thylakoid membrane of B. corticulans.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.

Characterization of the artificially covalent conjugate of B-phycoerythrin and R-phycocyanin and the phycobilisome from Porphyridium cruentum

B-phycoerythrin (BPE) and R-phycocyanin (RPC) were purified from Porphyridium cruentum by Sephadex G-200 chromatography, then the BPE was attached covalently to the RPC by reacting their amino groups to form the artificially covalent BPE-RPC conjugate in which the excitation energy can transfer from the BPE to the RPC with low efficiency. Meanwhile, the intact phycobilisome (PBS) consisting of BPE, RPC, APC and L-CM was isolated and purified from Porphyridium cruentum, and the purified PBS was found to keep intact if the solution contains sucrose. Comparison of spectroscopic properties between the purified PBS and the BPE-RPC conjugate suggests that the BPE-RPC conjugate is much more stable than the purified PBS. The construction of BPE-RPC conjugate with low efficiency of the excitation energy transfer may be useful for preparing phycobiliprotein probes. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro-particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 mug/mg soluble proteins on average and the highest value was 2.497 mug/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bio-reactor producing high value materials such as edible HBV vaccine was discussed as well.

Isolation, properties and spatial site analysis of gamma subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin and Porphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native gamma subunits were isolated from the reaction mixture. The process of degradation of phycoerythrin with proteinaseK showed that the gamma subunit is located in the central cavity of (alpha beta)(6) hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated gamma subunit showed that the absorption peaks of phycoerythrobilins on alpha or beta subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated gamma subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated gamma subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.

The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum

It is well known that invertebrates are devoid of adaptive immune components and rely primarily on innate immunity to defend against pathogens, but recent studies have demonstrated the existence of enhanced secondary immune protection in some invertebrates. in the present study, the cumulative mortality of scallops received two successive Listonella anguillarum stimulations was recorded, and variations of immune parameters including phagocytosis (phagocytic rate and phagocytic index), phenoloxidase-like enzyme, acid phosphatase and superoxide dismutase activities were also examined. The scallops received a previous short-term L anguillarum stimulation were protected against a long-term stimulation of L. anguillarum. Significantly higher level of phagocytic activities and acid phosphatase activity were observed in the scallops received twice stimulations compared with those only received the secondary stimulation. These results indicated that a short-term immersion with L. anguillarum modulated the scallops' immune system and endowed the scallops with enhanced resistance to the secondary bacterial stimulation: phagocytosis and acid phosphatase were suspected to be involved in the protection. (C) 2008 Elsevier Inc. All rights reserved.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32%similar to 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, A)Dnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana. (C) 2007 Published by Elsevier Inc.

Vertical distribution of bacterial and archaeal communities along discrete layers of a deep-sea cold sediment sample at the East Pacific Rise (similar to 13 degrees N)

The community structure and vertical distribution of prokaryotes in a deep-sea (ca. 3,191 m) cold sediment sample (ca. 43 cm long) collected at the East Pacific Rise (EPR) similar to 13 degrees N were studied with 16SrDNA-based molecular analyses. Total community DNA was extracted from each of four discrete layers EPRDS-1, -2, -3 and -4 (from top to bottom) and 16S rDNA were amplified by PCR. Cluster analysis of DGGE profiles revealed that the bacterial communities shifted sharply between EPRDS-1 and EPRDS-2 in similarity coefficient at merely 49%. Twenty-three sequences retrieved from DGGE bands fell into 11 groups based on BLAST and bootstrap analysis. The dominant groups in the bacterial communities were Chloroflexi, Gamma proteobacteria, Actinobacterium and unidentified bacteria, with their corresponding percentages varying along discrete layers. Pairwise Fst (F-statistics) values between the archaeal clone libraries indicated that the archaeal communities changed distinctly between EPRDS-2 and EPRDS-3. Sequences from the archaeal libraries were divided to eight groups. Crenarchaea Marine Group I (MGI) was prevalent in EPRDS-1 at 83%, while Uncultured Crenarchaea group II B (UCII B) abounded in EPRDS-4 at 61%. Our results revealed that the vertically stratified distribution of prokaryotic communities might be in response to the geochemical settings and suggested that the sampling area was influenced by hydrothermalism. The copresence of members related to hydrothermalism and cold deep-sea environments in the microbial community indicated that the area might be a transitional region from hydrothermal vents to cold deep-sea sediments.

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.

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.

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。

皱纹盘鲍的遗传育种与养殖技术研究

优良的种质是产业发展的重要保证，品种更新和养殖技术的发展已经给世界农业带来了令人瞩目的成就，然而我国水产生物的育种工作刚处于起步阶段，而育种技术的研究则更是滞后。借鉴陆生生物中发展起来的相对成熟的研究方法，可以帮助加快海洋生物遗传育种相关研究的进度。本研究以我国北方海区重要的海洋经济动物－皱纹盘鲍为研究对象，从表型遗传、数量性状遗传等2个方面开展了皱纹盘鲍的遗传育种研究，同时从幼鲍培育密度与分选效应等方面研究了皱纹盘鲍的中间培育技术。 主要结果如下： 1. 皱纹盘鲍的贝壳颜色遗传、食物对贝壳颜色表现型的影响，贝壳颜色与生长速度间的关系 将贝壳颜色为橘红色（O表型）的突变型皱纹盘鲍与贝壳颜色为绿色（G表型）的野生型皱纹盘鲍进行了连续2代的交配实验。结果表明：皱纹盘鲍橘红色的贝壳颜色相对于绿色的贝壳颜色为隐性性状，皱纹盘鲍的贝壳颜色表型受单位点、2个等位基因遗传控制，其中基因型为oo的个体，贝壳颜色的表现型为橘红色（O表型），而基因型为GG或Go的个体，贝壳颜色的表现型为野生型（G表型）。 为探讨食物类型对不同基因型皱纹盘鲍贝壳颜色表现型的影响，对不同贝壳颜色表型的个体投喂不同种类的食物，结果表明，除遗传因素外，皱纹盘鲍的贝壳颜色表现型显著地受食物类型的调控。其中oo基因型的个体，在摄食底栖硅藻（Navicula sp.）和红藻时，贝壳颜色的表型为橘红色；而在摄食褐藻、绿藻和以海带粉为唯一海藻源的人工配合饵料时，贝壳颜色的表型为黄色。GG和Go基因型的个体，在摄食底栖硅藻、红藻时，贝壳颜色的表型为褐红色；在摄食褐藻、绿藻和以海带粉为唯一海藻源的人工配合饵料时，贝壳颜色的表型为绿色。该结果表明，相同基因型的皱纹盘鲍在摄食不同类型的食物时，贝壳表现型不同，即不同类型的食物可以导致2种基因型皱纹盘鲍的贝壳颜色表现型在一定范围内发生转换：oo基因型的个体，贝壳的颜色可以表现为橘红色或者黄色，不会出现野生型皱纹盘鲍的褐红色或绿色；而GG与Go基因型的个体，相应的贝壳颜色表型只能是褐红色或者绿色，不会出现oo基因型可能表现的橘红色或黄色。特定基因型的皱纹盘鲍，在摄食特定类型的食物时贝壳的相应部位可表现出特定的颜色。皱纹盘鲍的这种“食物－贝壳颜色”的相关性可作为一种形态标记，用于标识皱纹盘鲍的个体和群体，该标记技术可用于皱纹盘鲍的养殖技术和遗传学研究。 此外，选用了贝壳颜色遗传学实验中建立的贝壳颜色发生分离的家系为实验材料，以壳长为指标，分析比较了来自相同家系的O表型与G表型个体之间的生长速度。结果表明，在幼鲍发育至412天止的3-5个统计时段内，没有在同一家系来源的2种贝壳颜色表型个体之间检验到生长速度的显著差异。 2. 皱纹盘鲍不同选育群体及杂交群体的贝壳形态参数分析 在皱纹盘鲍的7个群体中（包括已经对生长速度为指标进行了多代人工选育的群体4个、野生群体之间直接杂交繁育的杂交F1群体3个），测量了4-6龄成体样本的壳长（L）、壳宽（W）、壳高（H）和壳重（Sw），并计算了L/（L+W+H）、W/（L+W+H）、H/（L+W+H）和Sw/（L×W×H）等4个壳形态学参数。用方差分析方法（MANOVA、ANOVA）统计并比较了这些壳形态参数在皱纹盘鲍群体间的遗传变异。结果表明，4个壳形态参数在不同群体间变异系数分别为0.34、0.74、2.62和6.54，其中，H/（L+W+H）与Sw/（L×W×H）在各供试群体间均具有较高的多态性且差异达显著水平，表明这2个参数在不同群体间存在较高的遗传变异。由于在活体情况下无法测量壳重（Sw）性状，建议以参数H/（L+W+H）为指标对皱纹盘鲍贝壳形态（如壳型）等进行人工选择。 3. 皱纹盘鲍成体阶段生长性状的遗传参数估计 采用巢式设计，分析了成体阶段不同发育期皱纹盘鲍的壳长与生长速率的遗传力、不同发育期的壳长性状之间的遗传相关、以及不同发育期的生长速率之间的遗传相关，结果表明：（1）壳长遗传力在受精后第70 、130、320、320、380、490与550天的雄性组分估计值分别为0.161 ± 0.075、0.312 ± 0.131、0.326 ± 0.331、0.135 ± 0.228、0.153 ± 0.185和0.180 ± 0.106；雌亲组分估计分别为0.312 ± 0.172、0.699 ± 0.168、0.695 ± 0.168、0.977 ± 0.407、0.427 ± 0.195和0.449 ± 0.027。（2）生长速率遗传力在受精后第320~380天、490 ~ 550天，雄、雌组分估计值分别为0.080 ± 0.120（雄）、 0.210 ± 0.191（雌）以及0.299 ± 0.146（雄）、0.306± 0.148（雌）。雌亲组分的壳长遗传力和生长速率遗传力估计值较大且均达显著水平，表明皱纹盘鲍在成体阶段依然受母性效应的影响。成体阶段生长性状遗传力水平的估计对制定科学的皱纹盘鲍育种方案有指导意义。（3）雄亲组分估计的不同发育期（第390 ~ 550天）壳长间遗传相关为0.597 ~ 1.000，雌亲组分估计为0.589 ~ 1.177。由雄亲、雌亲组分估计，受精后第320~380天与第490 ~ 550天两个发育阶段生长速率间遗传相关均接近于0。雌亲组分估计不同发育期壳长间遗传相关均达显著水平（t0.05, d.f.=13 = 4.33 ~ 11.69，P<0.01），表明壳长性状早期选择有效，即在皱纹盘鲍早期阶段依据壳长性状对个体进行择优或去劣可在后期阶段获得壳长较大的个体。由于使用的雄亲数目少（8个父系半同胞），实验中以雄亲组分估计的遗传参数误差较大。 4. 皱纹盘鲍选育系间的群体杂交 进行了皱纹盘鲍4个人工选育系之间的完全双列杂交实验，以群体交配的方式共建立了16个组合；此外，以大连“98”选群与汕头“S”选群为亲本，以群体交配的方式建立了4个交配组合。对不同方向的杂交组合进行了中亲杂种优势、超亲杂种优势以及配合力等方面的评价。 （1）测量了4个选育群体（R、97、S和J）及其各杂交组合在受精后第9、20和30天时的壳长，统计分析了不同选育系间壳长性状的差异、评价了不同方向杂交组合的中亲与超亲杂种优势、以及配合力。结果如下： 选育系群体内交配繁育的4个组合，在受精后第9、20和30天的壳长均有显著差异，其中，97  97组合在早期发育各阶段均为最小，分别为0.462 ± 0.023mm、0.698 ± 0.057mm和1.476 ± 0.234mm；S  S组合的3次测量值均为最大，分别为0.522 ± 0.023mm、0.824 ± 0.084mm和1.798 ± 0.229mm。 两个方向杂交组合与选育系亲本群体内交配组合的平均值和高亲值比较，得到如下结果：（A）受精后第9天壳长表现正向中亲杂种优势的组合有6个、表现负向中亲杂种优势的组合6个，其中J  97组合的中亲优势率最高，为9.05%；R  S组合最低，为-6.61%。正向高亲杂种优势组合有4个、负向高亲杂种优势组合有8个，其中S  J组合的高亲优势率最高，为5.77%；R  S组合最低，为-7.96%。（B）受精后第20天壳长表现正向中亲杂种优势的组合有7个、表现负向中亲杂种优势的组合5个，其中J  97组合的中亲优势率最高，为12.60%；J  R组合最低，为-8.72%。正向高亲杂种优势组合有3个、负向高亲杂种优势组合有11个，其中J  97组合的高亲优势率最高，为12.20%；J  R组合最低，为-12.67%。（C）受精后第30天壳长表现正向中亲杂种优势的组合有7个、负向中亲杂种优势的组合5个，其中97  S组合的中亲优势率最高，为24.08%；S  97组合最低，为-12.69%。正向高亲杂种优势组合有6个、负向高亲杂种优势组合有6个，其中97  S组合的高亲优势率最高，为15.95%；S  J组合最低，为-19.44%。上述结果表明，皱纹盘鲍不同选育系之间的交配组合，杂种优势率差异很大，因此，通过组配实验，将杂种优势率高的交配组合选择出来应用于生产，可望显著提高目标性状的产量。 对早期发育阶段各生长期壳长性状，亲本一般配合力（GCA）、各杂交组合间特殊配合力（SCA）以及正反交（REC）效应值进行方差分析，结果表明：各亲本GCA差异显著，说明各选育群体存在显著的遗传差异，其中汕头选群“S”在测量的各个生长期均为正值且显著大于其它各亲本；特殊配合力（SCA）以及正反交（REC）效应值较大在各杂交组合间存在显著差异，说明在早期生长发育阶段非加性遗传效应（显性和上位效应）占主导地位。综合各个生长期亲本GCA和杂交组特殊配合力（SCA）以及正反交（REC）效应值，杂交组合97×S在早期生长阶段不仅有较高SCA值而且两个亲本也具有较大的GCA值，表明选育系97和S较适宜作为杂交亲本使用。 （2）大连“98”选群与汕头“S”选群进行2×2因子设计的群体杂交实验，比较了各交配组合早期存活相关性状如受精率、孵化率、变态率以及壳长性状，评价了两个方向杂交组合平均以及不同方向杂交组合的中亲杂种优势率。结果表明早期发育阶段各组合间的受精率无显著差异，而孵化率、变态率等两个杂交方向平均的中亲杂种优势率为5.49%与12.53%，高于壳长性状的优势率（0.936-1.534%）。方差分析结果表明不同方向的杂交组合在早期发育阶段存活相关性状以及壳长性状存在显著差异。孵化率、变态率性状，S×98的中亲杂种优势率分别为13.21%与21.10%，均高于98×S的-3.84%与3.85%；而第10和25d壳长性状，S×98的中亲杂种优势率为1.14%与-2.52%，低于98×S的1.93%与4.41%。 为进一步评价“98”选群与“S”选群不同交配组合在不同温度条件下的生长，进行了基因型与环境的互作研究。从“98”选群与“S”选群的4个交配组合中分别取5月龄幼鲍100头，各组合随机分成3组，每组1个重复，分别于12°C、16°C和 22°C温度条件下进行培育，比较各交配组合基因型与温度对幼鲍生长的影响。不同温度条件下，各组合壳长生长的方差分析结果表明，基因型和温度都能够对幼鲍生长以及最终壳长产生极显著的影响（P < 0. 01），它们的交互作用也达到显著水平（P < 0.05）。杂交子代的幼鲍壳长在12°C、16°C和 22°C温度条件下均表现出杂种优势，双向杂交的中亲杂种优势率分别为5.32%、5.55%和0.03%，表明低温条件（12°C），比高温条件（22°C）下有更强的杂种优势。汕头“S”选群的早期孵化率、变态率、生长性状以及低温条件下幼鲍生长性状的单亲杂种优势率分别为16.64%、42.49%、3.42~5.79%和5.73~9.15%，单亲杂种优势率较大，表明可通过杂交手段，显著地改良汕头“S”选群在早期发育阶段的生长速度、存活率以及幼鲍期的生长性状。本研究的结果支持了Lerner（1954）杂种优势的基因与环境互作学说。 5. 皱纹盘鲍幼鲍的中间培育技术研究 （1）对南方越冬方式的评价 目前，每年的11月前后，将6-7月龄幼鲍运往南方的闽东、闽中、闽南沿海越冬，翌年4月至6月再运回到北方（大连、山东半岛）的养殖模式已经普遍应用于皱纹盘鲍的实际生产，为评价南方越冬的幼鲍培育方式，本研究分别以不同幼鲍材料在闽东三都海湾进行了越冬培育实验。 选择生产上壳长分别为18.37 ± 1.28 mm、15.89 ± 1.10 mm、14.55 ± 1.10 mm与10.59 ± 0.84 mm的幼鲍进行了为期6.5个月的越冬培育，实验结束时，存活率分别为95.56 ± 2.21%、90.55 ± 1.96%、83.97 ± 1.63%与63.30 ± 2.79%。回归分析表明，供试幼鲍在实验起始时的壳长与越冬阶段的存活率成正相关（P = 0.018 < 0.05）。该结果表明，提高幼鲍的规格可显著提高皱纹盘鲍的越冬成活率，因此对于实际生产而言，采取适当措施提高皱纹盘鲍越冬苗种的规格将大幅增加生产的收益，而采用生长速率快的品种、品系或提早采苗均可实现该目标。综合各规格组幼鲍，幼鲍在南方开放性水域进行越冬培育的平均存活率较高，可达到91.38±0.01%，从幼鲍南方越冬的存活曲线可以看出，幼鲍的死亡主要集中在从大连运至福建某地后的15天内，出现死亡高峰的原因可能是由于运输过程的胁迫。此外，2月及4月中下旬水温出现显著降低或回升时也有较明显的死亡出现。该部分结果，对皱纹盘鲍幼鲍的养成管理有指导意义，可以通过合理安排越冬时间、避开死亡的敏感期等措施减少苗种越冬阶段的死亡量。 以中国大连野生群体繁育的子一代为亲本（10♀，10♂），以群体交配的方式繁育F2代个体为实验材料，分别于南方海区以及北方室内升温水方式下进行生长、存活比较，结果表明南方越冬培育方式下，幼鲍壳长的日增长率为81.37-108.89 µm•day-1，与北方室内升温培育条件相比，壳长生长提高了1.08 ~ 1.68倍；而存活率无显著差异。皱纹盘鲍幼鲍南方越冬方式的优势主要体现在鲍鱼幼鲍的生长速度加快，同时节约养殖场的能耗 （2）幼鲍培育过程中的养殖密度与分选效应评价 以3种规格皱纹盘鲍幼鲍为材料比较幼鲍在4个培育密度以及分选或混养条件下壳长的平均日生长及特定生长率。在南方越冬培育方式下实验进行106天，多因素方差分析结果表明实验初始幼鲍的壳长以及培育密度对壳长的生长有显著影响，而且密度效应在不同幼鲍起始规格组中有不同表现；分选没有能够提高不同规格组的生长。本研究的结果对皱纹盘鲍幼鲍的越冬培育有一定的指导作用。

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.