Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

Detection and quantitative analysis of polychlorinated biphenyls in tilapia from Hawaiian waters

The purpose of this study was to investigate polychlorinated biphenyls (PCBs) contamination in tilapia (Oreochromis mossambicus) collected from the Manna stream and Ala Wai Canal of O'ahu, an island of the geographically isolated Hawaiian archipelago. Our results show that the average concentrations of PCBs varied from 51.90 to 89.42 ng g(-1) lipid weight for the sampling sites. Relative toxic potencies (RTPs) and toxic equivalencies (TEQs) were determined to be 20.38-40.60 ng TCDD g(-1) lipid weight and 2.89-4.17 ng TEQ g(-1) lipid weight by 7-ethoxy-resorufin-O-deethylase (EROD) activity analysis and calculation of PCB concentrations based on toxic equivalency factors (TEFs), respectively. Penta-chlorinated congeners were found to be predominant, which revealed that Aroclor 1254 was a possible major source of PCBs in our fish samples. PCB 118, an indicator PCBs, constituted more than 55% and 30% of the total PCBs and TEQs, respectively. In addition, PCB 118 was found to have a linear correlation to the total PCBs (R = 0.975) and TEQs (R = 0.782). Detection of concentrated PCBs in Hawaiian waters suggests a potentially adverse impact of this pollutant on human health, as well as ecological systems, and suggests the necessity of environmental monitoring and hazard assessment of PCBs within the Hawaiian Islands. (c) 2008 Published by Elsevier Ltd.

光敏核不育水稻叶片蛋白质的双向电泳分析及苗期的光周期反应

光敏核不育水稻农垦58S由晚粳农垦58突变形成。具有在适宜温度条件下，长日照诱导雄性不育、短日照诱导雄性可育的基本特性。光敏核不育水稻育性转换机理的阐明是两系法杂交稻技术的关键。 1.克隆光敏不育基因是研究光敏核不育水稻育性转换机理的一个重要方面，本文对通过反映农垦58S和农垦58遗传背景差异的蛋白质或受光周期调节的蛋白质实现克隆光敏不育基因的策略进行了可行性研究，得到以下结果： (1).利用双向电泳技术在光敏核不育水稻是58S叶片中发现一个不存在于农垦58的蛋白质，其分子量为59.8kDa，等电点pH为5.9（称为Pa），该蛋白的存在不受光照条件、发育时期的影响，反映出农垦58S与农垦58遗传背景的差异。 (2).Pa蛋白与农垦58S叶绿体P61蛋白具有相同的分子量、等电点和N－端氨基酸顺序，在不同品种水稻中具有相同的分布，因此它们很可能是同一个蛋白质分子。 (3).利用双向电泳技术发现P61(Pa)和P41蛋白不仅存在于光敏不育系中，也存在于常规可育粳稻中，与光敏不育性状没有平行关系。 (4).利用双向电脉技术发现10天14小时长日照能在农垦58S和农垦58中诱导一个分子量为36kDa、等电点pH为5.2的蛋白质（称为P_b)，该蛋白的表达受光敏色素的调控。因此P61（Pa）、P41及P_b蛋白均与光敏不育性状无直接关系，推测克隆这些蛋白的基因无法直接获得光敏不育基因。 2.在育性转换光周期敏感期已经发现长日照使农垦58S叶绿体发育不良，但在苗期光周期敏感期内，目前尚不知长日照是否会有同样的效应。本文以光周期对农垦58S苗期叶绿体发育的影响为主要内容，研究了农垦58S苗期的光周期反应，得到以下结果： (1).农垦58S从5叶龄期至6叶龄期开始对光周期敏感，短日照开始能诱导茎尖分化幼穗。 (2).不同的光周期对农垦58S 4叶龄期新展叶片叶绿体发育的影响无明显差异，叶结体结构与功能均表现正常。 (3).不同的光周期对农垦58S 6叶龄期新展叶片叶绿体发育的影响有明显差异。与短日照相比，长日照引起农垦58S部分叶绿体发育不良，导致光化学活性减弱、超分子结构异常。长日光周期对农垦58S叶绿体发育的不良效应可能是光周期敏感期内存在的一种特殊现象。

I.小麦非叶器官碳酸酐酶活性与定位分析II.镉毒害导致芦苇胚性细胞死亡的激素调控研究

第一部分： 通过生理测定和化学染色分析了冬小麦品种小堰54和京411的叶片和非叶片组织的碳酸酐 酶活性。叶片碳酸酐酶活性(CA)在挑旗时期达到最大值，之后减少到最小，而在饱粒期又呈 增加趋势。从灌浆期到饱粒期，颖片和内稃的CA活性均减少，而外稃和种皮的CA活性均增加。在饱粒期，小堰54的叶片、颖片、外稃和种皮CA活性均高于京411。组织化学染色表明，CA主要分布在旗叶的叶肉细胞叶绿体中，也分布在非叶片组织颖片、外稃和内稃的叶肉和维管束鞘细胞的胞质中。这些结果表明，小麦非叶组织叶肉和维管束鞘细胞的胞质中的CA可能对饱粒期冬小麦的C4光合途径起作用。饱粒期小堰54的C02传递到Rubisco酶速率和抗旱性较京411高。 第二部分： 以继代培养的芦苇胚性细胞为材料，利用台盼兰拒染法检测了悬浮细胞死亡过程，并利用石蜡切片法及苏木精染色法观察了不同浓度镉对芦苇细胞的毒害作用。1000μM的CdCl2迅速导致芦苇悬浮细胞死亡，200μM的CdClz在接种后第5天引起悬浮细胞死亡，100μM的CdCl2在接种后第7天引起悬浮细胞死亡，≤50μM的CdClz在接种后7天不引起悬浮细胞死亡。同时对不同浓度镉处理的芦苇胚性细胞的内源植物激素和可溶性蛋白质进行分析，≤50μM的镉浓度显著地降低胚性细胞内IAA、ZR、GA3和GA4的含量，却提高ABA的含量，抑制可溶性蛋白质的合成：≥100μM的镉浓度显著地提高IAA、ZR、GA3和GA4的含量，却降低ABA的含量，促进可溶性蛋白质的合成。这些结果表明，镉的毒害至少包括镉浓度决定的两种细胞死亡机制，高浓度的镉(1000μM)引起的细胞死亡应当为坏死，而100μM的镉引起植物悬浮细胞发生程序性死亡。在较高浓度(≥100μM)的镉处理下，芦苇细胞内内源IAA、ZR、GA3和GA4的浓度较高，可能调控可溶性蛋白质的合成而促进细胞发生程序化死亡。

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Intergeneric conjugation in holomycin-producing marine Streptomyces sp strain M095

Marine Streptomyces are potential candidates for novel natural products and industrial catalysts. In order to set up biosynthesis approach for a holomycin-producing strain M095 isotated from Jiaozhou Bay, China, a genetic transformation system was established using intergeneric conjugation. The plasmid pIJ8600 consists of an origin of replication for Escherichia coli, a phage integrase directing efficient site-specific integration in bacterial chromosome, thiostrepton-induced promoter and an attP sequence. Using E. coli ET12567 (pUZ8002) carrying pIJ8600 as a conjugal donor, while it was mated with strain M095, pIJ8600 was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. The frequency of exconjugants was 1.9 +/- 0.13 x 10(-4) per recipient cell. Analysis of eight exconjugants showed pIJ8600 was stable integrated at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of growth and antimicrobial activity analysis indicated that the integration of pIJ8600 did not seem to affect the biosynthesis of antibiotics or other essential amino acids. To demonstrate the feasibility of above gene transfer system, the allophycocyanin gene (apc) from cyanobacterium Anacystis nidulans UTEX625 was expressed in strain M095, and the results indicated heterologous allophycocyanin could be expressed and folded effectively. (c) 2006 Elsevier GmbH. All rights reserved.

attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

海洋红藻多管藻和松节藻的化学成分及抗氧化活性研究

海洋生物具有产生丰富多样的次生代谢产物的能力，其中红藻门松节藻科海藻卤代次生代谢产物以其结构新颖、生物活性独特引起了天然产物化学家的重视。 本论文对海洋红藻多管藻和松节藻进行了化学成分研究，综合利用各种色谱学方法 (硅胶柱层析、反相硅胶柱层析、凝胶Sephadex LH-20柱层析、半制备高效液相色谱以及重结晶等) 和现代波谱学技术 (IR、UV、EI-MS、FAB-MS、HR-ESI-MS、CD、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、HMBC等)，共分离鉴定了100个化合物，发现25个新化合物。 从多管藻中分离鉴定38个化合物 (24个溴酚化合物)，其中7个新化合物 (均为溴酚化合物)，包括1个菲并呋喃结构溴酚 (P1), 2个二氢菲结构溴酚 (P2, P3)，1个含 5,7-dihydrodibenzo[c,e]oxepine 结构溴酚 (P4)和3个简单溴酚 (P5, P6, P7)。P1 (urceolatin) 属首例报道的具有菲并呋喃结构的天然产物，从该种中分离的化合物P12 和 P13 可能是其生源合成的前体。P2和P3为第二例报道的具有二氢菲结构的溴酚化合物。 从松节藻中分离并鉴定了62 个化合物，其中18 个为溴酚类新化合物，44 个为已知化合物。化合物具有多变的取代基团，包括2 个脲基吡咯烷酮溴酚化合物 (R1, R2), 4 个γ-脲基丁酸溴酚化合物 (R3-R6)，5 个酰胺溴酚化合物 (R7, R8, R9, R13, R14)，1 个溴酚砜化合物 (R12), 1 个Xanthene 溴酚化合物 (R10)和5 个简单溴酚化合物 (R11, R15, R16, R17, R18)。R1、R2 是首例报道的含有脲基吡咯烷酮片段的天然产物，R10 为首次报道的溴代Xanthene 类天然产物。 对分离到的化合物进行了清除DPPH 和ABTS两种自由基活性的筛选。结果发现溴酚类天然产物具有显著的DPPH自由基清除活性，其中R3 的IC50 仅为3.3 μM, 其活性强度约为阳性对照BHT (IC50 为82.1 μM) 的24倍。另外，溴酚类天然产物对ABTS自由基有较强的清除活性，R2 的TEAC（Trolox efficency activity capacity）值为5.2 mM，约为阳性对照 (ascorbic acid, 1.02 mM) 的 5 倍。初步的构效关系研究发现，稠环分子、多羟基和邻位甲氧基等结构特点能有效增强DPPH 自由基清除活性；特殊取代基如脲基、吡咯烷酮等含有氮原子的基团，能有效增强ABTS 自由基清除活性，多羟基、溴代等结构特点也使其活性有所增强。 本研究结果丰富了海藻卤代化合物的结构类型，为多管藻和松节藻的合理利用提供了一定的科学依据。

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3' untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5' flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. (c) 2005 Elsevier B.V All rights reserved.

Polyethyleneimine-Functionalized Platinum Nanoparticles with High Electrochemiluminescence Activity and Their Applications to Amplified Analysis of Biomolecules

Polyethyleneimine-functionalized platinum nanoparticles (PtNPs) with excellent electrochemiluminescence (ECL) properties were synthesized and applied to the amplified analysis of biomolecules. These particles were prepared at room temperature, with hyperbranched polyethyleneimine (HBPEI) as the stabilizer. The UV/Vis absorption spectra and transmission electron microscopy images clearly confirmed the formation of monodisperse PtNPs. Such particles proved to possess high stability against salt-induced aggregation, enabling them to be employed even under high-salt conditions. Owing to the existence of many tertiary amine groups, these particles exhibited excellent ECL behavior in the presence of tris(2.2'-bipyridyl)ruthenium(II). An HBPEI-coated particle possessed an ECL activity that was at least 60 times higher than that of a tripropylamine molecule. Furthermore, these particles could be immobilized on the 3-aminopropyltriethoxysilane-treated quartz substrates to amplify the binding sites for carboxyl groups. Through this approach, PtNPs were applied to the amplified analysis of the hemin/G-quadruplex DNAzyme by using the luminol/H2O2 chemiluminescence method.