利用荧光原位杂交技术（FISH）对中国明对虾和栉孔扇贝若干重要基因定位的研究

本研究将荧光原位杂交（Fluorescence in situ hybridization, FISH）技术引入中国明对虾和栉孔扇贝这两种重要的海水养殖动物的染色体研究中，建立了相关技术平台，在染色体上定位了部分功能基因，详述如下： 1. 在中国明对虾和栉孔扇贝中建立了FISH平台，利用单色和双色FISH技术在中国明对虾和栉孔扇贝染色体上成功定位了多拷贝基因，发现5S rDNA定位于中国明对虾的一对同源染色体上，栉孔扇贝18S rDNA和组蛋白序列也各自定位于一对同源染色体上，它们均可以作为染色体特异性探针来鉴别染色体。 2. 在栉孔扇贝中发展了BAC-FISH技术，定位了包含热休克蛋白70（HSP70）、丝氨酸蛋白酶（Serine Protease）和脂多糖葡聚糖结合蛋白（LGBP）等免疫相关基因的BAC克隆，发现它们均定位于一对同源染色体的长臂上。利用双色BAC-FISH技术，发现6个包含栉孔扇贝LGBP基因的BAC克隆在间期细胞核上共定位。对栉孔扇贝5个探针进行了同时定位，初步鉴别了栉孔扇贝5条染色体。 3. 在栉孔扇贝热休克蛋白70（HSP70）、丝氨酸蛋白酶（Serine Protease）和脂多糖葡聚糖结合蛋白（LGBP）等免疫相关基因内部发掘了数个潜在的SNP位点，展示了栉孔扇贝SNP位点的丰富性。这些SNP位点可以用于图谱整合。

Effects of intraperitoneal injection of cortisol on nonspecific immune functions of Ctenopharyngodon idella

After grass carps Ctenopharyngodon idellus were injected with cortisol, with (CBC) and without (C) a cocoa butter carrier, the effects of both slowly and rapidly acting exogenous cortisol oil their non-specific immune functions were investigated. On the one hand, after injection with CBC, the cortisol concentration and lysozyme activity in fish serum were enhanced and were sustained at high levels for a long period (30 days). The killing activity in the serum declined with time, and phagocytosis of head kidney macrophages diminished significantly (P < 0.05 or P < 0.01). The leukocrit values in the high dose group (31-8 mg cortisol fish(-1)) increased over time, however, with the maximum average being 5.6% at day 30. The spleen mass index in the high dose group was 0.93 x 10(-3) after 30 days, notably lower (P < 0.05) than that in the control group. In addition, a decrease in resistance to Aeronionas hydrophilo infection in cortisol-treated fish was shown, with the final cumulative mortalities being 54.5 and 66.7% in the low and high dose groups, respectively. On the other hand, there was a decrease in both serum cortisol concentration and lysozyme activity of the experimental fish within 2 weeks after injection with C, where plasma bactericidal activities in the high dose group (31-8 mg cortisol fish(-1)) were remarkably lower (P < 0.01) than those in the control group at each sampling, but were increased slightly over time. The results of which were different from those in the CBC trial. Phagocytic activity of head kidney macrophages and spleen mass index decreased significantly (P < 0.05), while there were increases in leukocrit value and cumulative mortality due to A. hydrophila. The results of which were similar to those in the CBC trial. This study indicated that the injection of cortisol depressed the non-specific immune functions of the grass carp and increased its susceptibility to disease. (c) 2005 The Fisheries Society of the British Isles.

构筑湿地部分亚硝化－厌氧氨氧化自养脱氮研究

废水部分亚硝化—厌氧氨氧化自养脱氮研究是目前废水生物脱氮领域研究的热点。为了开发低浓度小城镇废水构筑湿地可持续自养脱氮新工艺，提高脱氮效率，减少占地面积，论文在对构筑湿地自养脱氮前置部分工作性能进行研究的基础上，在法国东部Evieu构筑湿地污水处理场对传统脱氮工艺及改进用于部分亚硝化—厌氧氨氧化脱氮的工艺进行了对比实验研究。 研究结果表明，采用改进的非饱和层(25cm)与饱和层(55cm)结合的湿地床与60cm深度的水平流构筑湿地床组合工艺，进行着完全不同于传统垂直流构筑湿地硝化与水平流构筑湿地反硝化的脱氮反应。氮平衡分析表明，这一脱氮过程是以厌氧氨氧化反应为主的脱氮反应。经分子生物学荧光免疫原位杂交（Fluorescent in situ hybridization, FISH）技术鉴定，证明了传统的垂直流构筑湿地以好氧氨氧化细菌为主，但厌氧氨氧化细菌与之共存；而在改进的湿地床及后续水平流湿地床低氧环境则是以厌氧氨氧化细菌为主。论文首次为构筑湿地系统在自然条件下通过合理设计实现厌氧氨氧化自养脱氮提供了可行的实验证据。实验研究得出如下主要结论： 1. 构筑湿地可通过合理设计促进自养脱氮反应实现；传统硝化反硝化脱氮工艺VF1-1+HF3-2总氮平均去除率为66.3%，VF1-3+HF3-2总氮平均去除率为59.4%，而改进后的VF1-2+HF3-2总氮平均去除率为71.0%；含有厌氧氨氧化自养脱氮反应的工艺提高了总氮去除率； 2. 构筑湿地自养脱氮反应过程不排斥异养反硝化作用存在，两者协同进行脱氮，脱氮效果稳定；而全程硝化的出水在水平流构筑湿地进行传统反硝化脱氮的同时，会存在异化性硝酸盐还原作用，使出水的氨氮浓度增加； 3. 构筑湿地具有较好的蓄积培养厌氧氨氧化细菌的能力，在19.7℃的进水水温条件下启动反应装置，运行培养100天即观察到所需要的生物体颜色呈棕红色；调整pH至适宜厌氧氨氧化反应的范围6.81-7.18；控制氧化还原电位Eh值在-148.34至120.18mV；反应装置进水的氨饱和，并且稳定进水水力负荷在构筑湿地污染物去除能力范围内以及为避免外环境因素如降雨量过大等对反应装置造成的冲击，设置缓冲装置等，是构筑湿地实现自养脱氮主要控制的工艺参数； 4. 水力停留时间设计为5天的水平流构筑湿地HF3-3更适合传统反硝化脱氮工艺；水力停留时间的延长并不能使厌氧氨氧化工艺进一步提高脱氮效率，采用厌氧氨氧化自养脱氮工艺为减少构筑湿地占地面积提供了可能。

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Gene structure and transcription of IRF-1 and IRF-7 in the mandarin fish Siniperca chuatsi

The genes of IRF-1 and IRF-7 have been cloned from the mandarin fish (Siniperca chuatsi). The IRF-1 gene has 4919 nucleotides (nt) and contains 10exons and 9introns, with an open reading frame (ORF) of 903 ntencoding301 aa. The IRF-7 gene has 6057 nt and also contains 10exons and 9introns, with an ORF of 1308 nt encoding 436 aa. The IRF-1 and IRF-7 genes have only one copy each in the genome. The transcription of IRF-1 and IRF-7 in different organs was analyzed by real-time PCR, and both molecules were constitutively expressed. The IRF-I and IRF-7 mRNAs were abundant in gill, spleen, kidney and pronephros. The temporal transcriptional changes for IRF-1, IRF-7 and Mx were investigated within 48 h after poly I: C stimulation in liver, gill, spleen and pronephros. An increased transcription was detected for IRF-1 and IRF-7 12 h post-stimulation, being earlier than the transcription of Mx protein; however, IRF-1 and IRF-7 transcription decreased while the Mx protein was stable at 48 h post-stimulation. (c) 2007 Published by Elsevier B.V.

Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a vitally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx wash also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Genetic analysis of all-fish growth hormone gene transferred carp (Cyprinus carpio L.) and its F-1 generation

Recombinant "all-fish" growth hormone gene (GH) was microinjected Into the fertilized eggs of carp. A comparison between the growth traits of transgenics and non-transgenics was carried out, and the transgenic individuals with significant "fast-growing" effect were successfully gained. A comparison on the reproductivities was also given out between the transgenics and their non-transgenic siblings, and showed that the reproductive capacity of transgenics was substantially equivalent to those of the non-transgenics. On the other hand, the genetic separation and the characteristic distribution of the F-1 generation were genetically analyzed, which gave solid evidence for the hypothesis that 2-3 chromosomes are integrated with transgene. In addition, the distinct biological effects for multisite-integrated transgenes were further discussed. The present study opens a door for the breeding of "fast-growing" transgenic fish.