Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.

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.

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Isolation of PSI from a siphonous marine green alga, Codium fragile

Three different forms of PS I complexes were isolated from a siphonous marine green alga, Codium fragile, by Triton X-100 sucrose gradient centrifugation. Zone III had a Chl a/b>20, and designated as PS I. core complex CC I because it created only CP I band in mild PAGE. Zone IV and V had absorption at 436 and 674 nm, 467 and 650 nm, and 540 nm, suggesting the presence of Chl a, Chl b, siphonaxanthin and siphonein, Chl a/b were 3.23 and 2.4, respectively. Both CP I and CP I a bands were observed when they were subjected to mild PAGE. Therefore, Zone IV and V were different forms of PS I complexes that consisted of CC I and different amount of light-harvesting complex LHC I. Zone III contained only 66 and 56 ku peptides in SDS-PAGE, while Zone IV and V had 4 different LHC I peptides of 25, 26, 26.2 and 27.5 ku in addition to 66, 56 ku peptides. Fluorescence emission spectra showed that efficient energy transfer were kept among pigments in isolated PS I complexes. Excitation energy absorbed by Chl b, siphonaxanthin and siphonein can be transferred to Chl a.

Isolation and nomenclature of pigment-protein complexes from the brown alga (Undaria pinnatifida Harv.)

Eight kinds of pigment-protein complexes were resolved from the thylakoid membrane of the brown alga (Undaria pinnatifida Harv.) by using non-ionic detergent decanoyl-N-methylglucamide and PAGE technique. According to the apparent molecular weights, spectra characteristics, polypeptide compositions and referring to the higher plant spinach, eight pigment-protein complexes were named under Anderson's terminology system as CP I a, CP I, CPa, LHC1, LHC2, LHC3, LHC4, LHC5.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

条斑紫菜孢子体与配子体PSII的特性以及单颗粒分析

选择条斑紫菜（Porphyra yezoensis）两个不同发育阶段（孢子体和配子体）作为研究对象，分别从孢子体与配子体的类囊体膜上分离到具有高放氧活性的PSII复合物。配子体PSII复合物的放氧活性为2269.77±152.94 μmolO2/chl(mg).h，孢子体PSII复合物的放氧活性为2256.33±141.81 μmolO2/chl(mg).h。对分离到的PSII复合物的稳定性和放氧活性进行了研究，结果表明：孢子体和配子体的PSII复合物，在4℃条件下保存比-80℃下放氧活性高，稳定性高。配子体跟孢子体比较，在-80℃保存条件下第六天就已经没有放氧活性，而此时-80℃下孢子体PSII复合物仍然具有放氧活性。同时对4℃下保存的PSII复合物进行分子筛柱层析，室温吸收光谱测定以及放氧活性测定，发现随着放氧活性逐渐降低的同时，蛋白大分子有聚合现象。室温吸收光谱表明经过长期的保存，吸收峰向短波长方向偏移，同时叶绿素易降解成为脱镁叶绿素。分别测定了配子体与孢子体PSII复合物的SDS-PAGE电泳蛋白条带的氨基酸序列，配子体PSII复合物经过SDS-PAGE电泳后的蛋白条带，经过质谱测定，鉴定出CP-47、D2、D1蛋白。孢子体PSII复合物，经过SDS-PAGE电泳后的蛋白条带，经过质谱分析鉴定出CP-47、CP-43、D2、D1蛋白。CP-47， CP-43蛋为PSII反应中心叶绿素P680的脱辅基蛋白，D1 与D2蛋白为PSII的反应中心蛋白。对纯化的PSII复合物，并首次采用单颗粒技术分析比较其形态结构，电镜观察证实纯化的PSII复合物为二聚体，颗粒大小约为13nm×7.8nm，经过计算总分子量约为500kDa，但是孢子体与配子体的PSII复合物在形态上有轻微差别，配子PSII复合物要略宽于孢子体复合物，孢子体PSII复合物周边棱角形较配子体明显。

东太平洋和中太平洋海盆铁锰结核的物质组成、成矿介质和成矿作用研究

本文在区域背景分析的基础上，详细研究了中太平洋CP区（7°-12°N，176°E-178°W）和东太平洋CC区（7°-14°N，138°-152°W）铁锰结核的铁锰矿物组成、化学成分、成矿介质特征及其形成控制因素，结果表明：铁锰结核内部构造具有几乎由纯的铁锰氧化物组成的平行纹层状构造到含有较多碎屑矿物粘土矿物的斑杂状构造的连续过渡特征；铁锰结核中的锰矿物主要是δ-MnO_2、钡锰锰矿和钠水锰矿，且以前两者为主，在以平行纹层状构造为主的结核中富δ-MnO_2、贫钡镁锰锰，δ-MnO_2/钡镁锰矿比值大于10，而在以斑杂状构造为主的结核中δ-MnO_2/钡镁锰矿的比值在1左右，铁矿物主要是针铁矿、四方纤铁矿等；铁锰结核中的化学元素可以分成五个具有独立成因意义的组合：锰元素组、铁元素组、造岩元素组、生物成因元素组和易溶盐类组分，具平行纹层状构造富含δ-MnO_2的结核中富集铁组元素，具斑杂状构造富含钡锰锰矿的结核富集锰组元素和造岩元素；铁锰结核中稀土元素含量是正常深海沉积物的2-3倍。水成型结核和混合成岩型结核上部稀土元素更富且具Ce的明显正异常，而成岩型结核和混合成因结核的下部稀土元素含量相对低，Ce的正异常也不明显，有时出现负异常；铁锰结核的直接成矿介质是大洋底层水和孔隙水，因此底层水和孔隙的物理化学条件就是决定结核地球化学特征的关键条件，影响大洋底层水、孔隙水物理化学条件变化的地质因素主要有南极底流、沉积速率、生物生产力等等；铁锰结核的生长是一非线性振荡式的地质地球化学作用过程。

西菲律宾海有机悬浮颗粒分布变化及其光衰减研究

论文以2004年在西菲律宾海获取的光学剖面资料为基础，首次将光学衰减系数作为悬浮有机颗粒的替代指标，对西菲律宾海上层水体悬浮颗粒及颗粒有机碳的时空分布和变化进行了研究。结果表明，光衰减系数cp同颗粒有机碳浓度具有显著的线性相关关系，这对于海洋初级生产力估算和碳循环研究具有重要意义。断面WE和SN上的浮游植物现存量与颗粒有机碳现存量变化趋势完全一致。上层水体中的颗粒有机碳主要来源于有机碎屑颗粒。西菲律宾海混合层的昼夜变化对于表层水体光学变异以及颗粒输出作用具有重要影响。表层水温、光衰减系数cp和混合水深都与海面光合有效辐射同相变化。表层水体中的颗粒有机碳现存量同混合水深同步变化，受混合层控制作用显著；而对于深层水体，推测主要受生物垂向迁移和颗粒聚集沉降的影响。 根据表层水体中微微型自养颗粒的计数分析结果，原绿球藻的丰度最高，其次为聚球藻，自养真核细胞的丰度最低。首次估算了三种微微型颗粒的散射系数，并与光衰减系数cp做了对比分析。研究表明，菲律宾海三种微微型颗粒的散射作用仅占光衰减系数cp的5%，其中原绿球藻、自养真核细胞和聚球藻分别占2.17%、2.85%和0.33%。根据微微型颗粒的散射特征，可大致将研究海区划分为三个生物水文学区域。 对菲律宾海上层水体的光场特征作了分析，结果表明水下辐照度Ed按照指数规律衰减。通过对漫衰减系数Kd的研究，首次证实了西菲律宾海非藻组分的光衰减作用占主要地位。表层水体中非藻组分与浮游植物的光衰减作用大小相近，随着水深增大，非藻组分的光衰减作用逐渐占据主导地位。此外，西菲律宾海离水辐亮度Lwn(555)和海面遥感反射率Rsr(555)同生物地球化学核心变量POC之间存在显著的指数相关关系，这对于海洋初级生产力的遥感估算和应用具有重要意义。

In vitro determination of antioxidant activity of proteins from jellyfish Rhopilema esculentum

In this study, the antioxidant activity of proteins isolated from jellyfish, Rhopilema esculentum Kishinouye (R. esculentum), was determined by various antioxidant assays, including superoxide anion radical-scavenging, hydroxyl radical-scavenging, total antioxidant activity, reducing power and metal chelating activity. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol, vitamin C and mannitol were used as standards in those various antioxidant activities. The crude protein (CP) and the protein fractions isolated by Sephadex chromatography, first peak (FP) and second peak (SP), had very low reductive power and metal chelating abilities compared to EDTA, but they showed strong scavenging effects on the superoxide anion radical, hydroxyl radical and varying total antioxidant activity. FP and SP exhibited stronger scavenging effects on the superoxide anion radical than BHA, BHT or a-tocopherol. The EC50 values of FP and SP were 6.12 and 0.88 mu g/ml, respectively, while values EC50 of BHA, BHT and alpha-tocopherol were 31, 61 and 88 mu g/ml, respectively. CP, FP and SP showed far higher hydroxyl radical-scavenging activities than did vitamin C or mannitol. The EC50 values of CP, FP and SP were 48.76, 45.42 and 1.52 mu g/ml, but EC50 values of vitamin C and mannitol were 1907 and 4536 mu g/ml, respectively. In a beta-carotene-linoleate system, SP and CP showed antioxidant activity, but lower than BHA. Of the three samples, SP had the strongest antioxidant activity. So, SP may have a use as a possible supplement in the food and pharmaceutical industries. (c) 2005 Elsevier Ltd. All rights reserved.

The synthesis and antioxidant activity of the Schiff bases of chitosan and carboxymethyl chitosan

Five kinds of Schiff bases of chitosan and carboxymethyl chitosan (CMCTS) have been prepared according to a previous method and the antioxidant activity was studied using an established system, such as superoxide and hydroxyl radical scavenging. Obvious differences between the Schiff bases of chitosan and CMCTS were observed, which might be related to contents of the active hydroxyl and amino groups in the molecular chains. (c) 2005 Elsevier Ltd. All rights reserved.

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn2+, Mg2+, and Mn2+ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn2+ had the best effects among the three metal cations and the effect was about 20 times of that of Zn2+ or Mg2+ and its maximal protease activity was 2.3 x 10(5) U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively. (c) 2005 Elsevier Ltd. All rights reserved.

Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro

The antioxidant potency of different molecular weight (DMW) chitosan and sulfated chitosan derivatives was investigated employing various established in vitro systems, such as superoxide (O-2(.-))/hydroxyl ((OH)-O-.) radicals scavenging, reducing power, iron ion chelating. As expected, we obtained several satisfying results, as follows: Firstly, low molecular weight chitosan had stronger scavenging effect on O-2(.-) and (OH)-O-. than high molecular weight chitosan. For example the O-2(.-) scavenging activity of low molecular weight chitosan (9 kDa) and high molecular weight chitosan (760 kDa) were 85.86 % and 35.50 % at 1.6 mg/mL, respectively. Secondly, comparing with DMW chitosan, DMW sulfated chitosans had the stronger inhibition effect on 0(2)(.-). At 0.05 mg/mL, the scavenging activity on O-2(.-) reached 86.26 %, for low molecular weight chitosan sulfate (9 kDa), but that of low molecular weight chitosan (9 kDa) was 85.86 % at 1.6 mg/mL. As concerning chitosan and sulfated chitosan of the same molecular weight, scavenging activities of sulfated chitosan on superoxide and hydroxyl radicals were more pronounced than that of chitosan. Thirdly, low molecular weight chitosan sulfate had more effective scavenging activity on 02 and (OH)-O-. than that of high molecular weight chitosan sulfate. Fourthly, DMW chitosans and sulfated chitosans were efficient in the reducing power, especially LCTS. Their orders were found to be LCTS > CTS4 > HCTS > CTS3 > CTS2 > CTS1 > CTS. Fifthly, CTS4 showed more considerable ferrous ion-chelating potency than others. Finally, the scavenging rate and reducing power of DMW chitosan and sulfated derivatives increased with their increasing concentration. Moreover, change of DMW sulfated chitosans was the most pronounced within the experimental concentration. However, chelating effect of DMW chitosans were not concentration dependent except for CTS4 and CTS1. (C) 2004 Elsevier Ltd. All rights reserved.

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology. (C) 2005 Elsevier Ltd. All rights reserved.