微囊藻毒素去毒酶转基因模型的构建

根据已克隆的鲢鱼(Hypophthalmichthysmolitrix)微囊藻毒素去毒酶cDNA全序列设计特异引物,利用PCR方法获得鲢鱼微囊藻毒素去毒酶基因编码区,将该编码区与绿色荧光蛋白连接,分别构建融合表达载体pEGFP-N1-sGST和双顺反子表达载体pIRES2-EGFP-sGST。利用脂质体转染法将融合表达载体pEGFP-N1-sGST转染Hela细胞,60h后检测到绿色荧光基因表达;通过显微注射,将双顺反子表达载体pIRES2-EGFP-sGST注入斑马鱼(Daniorerio)受精卵,获得了转鲢鱼微囊藻毒素去毒酶基因斑马鱼,从而构建了微囊藻毒素去毒酶转基因模型。上述2种转基因模型的成功构建为进一步研究鲢鱼、鳙鱼(Aristichthysnobilis)、罗非鱼(Oreochromisnilotica)等淡水鱼类微囊藻毒素去毒酶基因调控元件、去毒分子机理及研发转基因鲢鱼、鳙鱼、罗非鱼等微囊藻毒素高效生物去毒器奠定了基础。     

水生生物对微囊藻毒素去毒分子机理及调控因子研究

近年来,随着工农业的不断发展,排入水体的各种污染物不断增加,加速了淡水水域的富营养化进程,使得浮游藻类大量繁殖,有害藻类水华频繁发生,世界各地不断有藻类污染水体引起人畜患病甚至死亡的事件报道。在淡水藻类中,毒性最强、污染范围最广的为蓝藻门,[第一段]     

微囊藻毒素对鱼类毒性影响的研究进展

微囊藻毒素 (Microcystins,简称MC)是一些有毒蓝藻,如微囊藻、颤藻和鱼腥藻等,产生的一种具有肝毒性的环状七肽。     

微囊藻毒素对鱼类的毒性效应

湖泊富营养化导致的蓝藻水华已成为国内外普遍关注的环境问题,它所带来的主要危害之一是产生的藻毒素对鱼类的影响。在已发现的藻毒素中,微囊藻毒素(microcystins,MCs)的分布广、毒性大、危害严重,而备受关注。阐述了MCs对鱼类的影响。微囊藻毒素能干扰胚胎的发育,降低孵化率,增加畸形率,影响存活率,胚胎孵化受微囊藻毒素影响还具有剂量依赖效应;野外室内实验均表明鱼类暴露于微囊藻毒素后不仅可在肝脏中富集还可在肌肉、肠道等组织器官中快速积累;对鱼类进行组织病理检测发现MCs可导致肝脏、肾脏、心脏、脑、鳃等组织受损;MCs在鱼体中的解毒过程可能开始于由谷胱甘肽S-转移酶催化的还原型谷胱甘肽的结合反应;MCs还可影响鱼类的生长、行为和血清生化指标,此外,还具有一定的免疫毒性。MCs的转运机制和分子作用机制以及在食物链中传递过程中对人类造成的潜在影响可能成为今后研究重点。     

肉毒杆菌二元毒素和细胞外酶

对肉毒中毒和肉毒毒蛋白的研究已有近百年历史,肉毒杆菌神经毒素（ＢｏＮＴ）作用机制研究的进展已使它成分为分析神经递质释放的胞吐分子机制和用于临床治疗的有用工具。近年发现,肉毒梭状芽胞杆菌Ｃ和Ｄ型菌株除分泌ＢｏＮＴ外,尚合成另外两类与ＢｏＮＴ结构和生物活必完全不同的蛋白质,肉毒二元毒素和肉毒细胞外酶,它们分别影响细胞骨架和细胞的生长发育,也成为分析相关生命过程的有效工具药,本文综述了这两类蛋白质的有关     

微囊藻毒素降解酶基因cDNA部分序列的克隆及检测分析

微囊藻毒素降解酶 (Microcystinase, MlrA) 是在自然环境中微囊藻毒素 (Microcystin, MC) 降解过程中起重要作用的酶。通过PCR方法从水体中扩增MlrA基因cDNA部分序列,序列长342 bp,编码113个氨基酸 (GenBank号:FJ972202),PCR方法系统性检测不同季节环境中水体、鱼体MlrA的存在情况。结果表明,克隆的序列与日本学者Saito所公布的 Sphingomonas sp. MD-1、Y2菌株的MlrA基因同源性高达96%和89%,与挪威 Sphingomonas sp. ACM-3962、澳大利亚 Sphingopyxis sp. LH21菌株的MlrA基因同源性分别为95%和94%,确定所获为鞘氨醇单胞菌菌株中的MlrA基因cDNA部分序列。同时对不同季节环境水体和部分鱼体MlrA基因的检测时,发现在水华发生的四月及五月,水体和所检测的鱼体中MlrA基因结果显示为阳性,而所检测的其余6个月份的全部水样以及7月和10月检测的鱼样,结果均呈阴性。     

微囊藻毒素合成酶基因的PCR检测方法

针对微囊藻毒素合成酶基因簇的核酸序列,筛选特异性引物,探索一种适用于自然水样中微囊藻产毒潜能检测的全细胞PCR方法。经灵敏度测试表明,这种PCR方法的检测下限相当于100cells。该方法不需要提取基因组DNA,检测所需水样量少,具有操作简便、快速、成本低、灵敏度高等优点,能应用于水库等饮用水源水体中具有产毒潜能的微囊藻的检测。     

微囊藻毒素对尼罗罗非鱼原代肝细胞致毒机理的探讨

采用离体细胞培养诱导方法,研究微囊藻毒素-LR(microcystin-LR,MC-LR)对尼罗罗非鱼(Oreochromis niloticus)原代肝细胞的毒性效应.尼罗罗非鱼原代肝细胞经10、50、150、500 μg/L MC-LR体外诱导24h后,单细胞微量凝胶电泳(SCGE)检测显示,与对照组相比处理组出现明显的彗星拖尾现象,说明MC-LR可引起尼罗罗非鱼肝细胞DNA的损伤,并随着剂量的增加,DNA的损伤程度增大.PI/Annexin V双染色流式细胞仪(FCM)检测表明MC-LR能明显引起肝细胞凋亡,与SCGE结果一致,且DNA损伤程度越大,细胞早期凋亡率越高,呈现明显的时间、剂量依赖性.本研究为进一步从分子、细胞水平阐明MC-LR的毒性以及致毒机理提供重要的理论依据.     

微囊藻毒素分子致毒机理研究近况

目前,日趋严重的水体富营养化使水华发生已成为全球性环境问题,产生水华的藻类因而也受到越来越多的重视。海洋赤潮中甲藻所产生的毒素已引起人类的严重的中毒事件,虽然淡水水华还未发现引起人类急性中毒事件[1,2],但由于饮用发生水华水体的水而导致家畜、家禽及野生动物死亡的事件频繁发生[3,4],淡水水华已引起科学家的关注。     

罗非鱼微囊藻毒素去毒相关基因克隆与活体表达研究

可溶性谷胱甘肽S-转移酶(Soluble glutathione S-transferase,sGST)催化微囊藻毒素(Microcystins,MCs)与还原型谷胱甘肽(GSH)的加合去毒代谢过程,谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)为sGST的去毒反应提供GSH,解偶联蛋白2(Uncoupling protein 2,UCP2)则可抑制微囊藻毒素诱发活性氧导致的肝细胞凋亡。本研究从罗非鱼肝脏通过简并引物克隆sGST、GPX与UCP2基因cDNA核心序列,并应用5’RACE和3’RACE技术分别扩增罗非鱼肝脏sGST基因cDNA序列5’末端和3’末端序列而获得其cDNA全序列。罗非鱼肝脏sGST基因cDNA全序列长861 bp,其中5’非翻译区(5-’UTR)为25 bp,3’非翻译区(3-’UTR)为167 bp,开放阅读框(ORF)为669 bp,编码222个氨基酸,包含脊椎动物完整sGST的2个功能域:N-末端功能域(GSH结合位点)和C-末端功能域(底物结合位点)。罗非鱼sGST与真鲷、条石鲷(Oplegnathus fasciatus)、斑马鱼同源性较高,达到64.3%—78.5%,而与人、大鼠、小鼠、牛、猪、鸡差异较大,氨基酸同源性为48.2%—55.9%。罗非鱼肝脏GPX、UCP2基因cDNA核心序列长280 bp、776 bp,分别编码92、258个氨基酸。罗非鱼GPX与条石鲷、虹鳟、斑马鱼、人、大鼠、小鼠、牛、猪GPX同源性均较高,达到69.6%—85.9%。罗非鱼UCP2与真鲷、斑马鱼、鲤鱼、欧洲白鲑(Leuciscus cephalus)、草鱼、人、大鼠、小鼠UCP2同源性更高,达到71.8%—93.8%。通过对罗非鱼(5—8 g)活体腹腔注射亚致死量MC-LR(50μg/kg bwt),发现微囊藻毒素对罗非鱼肝脏sGST基因表达有显著的诱导作用(p<0.05),注射微囊藻毒素24h后sGST基因mRNA表达水平上调80%。注射微囊藻毒素24h后,虽然罗非鱼肝脏GPX与UCP2基因mRNA表达水平亦出现明显的升高趋势,但两者均未出现显著性的变化(p>0.05)。本研究从基因表达调控的角度证实,罗非鱼肝脏sGST在微囊藻毒素去毒过程中可能发挥关键作用,同时也说明罗非鱼肝脏GPX、UCP2基因可能在微囊藻去毒过程中发挥协同作用。     

Zoogeography and biodiversity of the freshwater fishes of Southeast Asia

The ichthyofauna of the freshwater system of Southeast Asia is extremely diverse. A recent estimate of about 1000 species is probably an understatement. More than 10 new species are being added to the list annually. The distribution pattern of the Southeast Asian freshwater fishes can be divided into five zoogeographic regions. The first one is the Salween basin in Burma, with fishes mainly of the Indian subcontinent origin such as Amblypharyngodon atkinsoni, Bangana almorae and Brachydanio jayarami. The second zoogeographic area is the Mekong, Chao Phraya and Mae Khlong drainages which harbour fishes typical of the mainland of Southeast Asia such as Acanthorhodeus deignani, Barbichthys nitidus and Cirrhinus siamensis. The Malay Peninsula is the third region whose species composition is heavily influenced by the Siamese (such as Homaloptera smithi, Tuberoschistura baenzigeri and Botia beauforti) and Indonesian (such as Botia hymenophysa, Luciocephalus pulcher and Parosphromenus deissneri) elements. The islands of Sumatra, Borneo and Java are the fourth zoogeographic area of fish distribution. These islands show a high degree of endemism, especially in fishes of the family Belontiidae. Finally, the freshwater system of the Philippines is the last zoogeographic region of Southeast Asia. The area is characterized by the presence of closely related species of the cyprinids especially in Lake Lanao. Currently, high diversity of these freshwater fishes is being threatened by land development, such as deforestation, road construction and land expansion for plantation. Recent studies in the Gombak River basin show the extermination of 41 per cent of native fish species from 1969 to 1990. This is probably due to the construction of highways, logging, as well as land clearing for agriculture.     

Response to De Silva et al. (2007): Endemic freshwater finfish of Asia: distribution and conservation status

De Silva et al . (2007) present an overview of the distribution and conservation status of the endemic freshwater fish of Asia. Within that review they use data from the IUCN Red List of Threatened Species™ (2006) to conduct an analysis of the conservation status of those endemic fish species. Their analysis is incorrect and provides a very misleading impression of the level of threat to Asian freshwater fish and to freshwater fish at the global scale. The errors stem from a misinterpretation of the data presented on the IUCN Red List. The sources of errors are discussed below and the opportunity is taken to clarify what the information on the IUCN Red List represents.     

The Distribution and Origins of Freshwater Fishes in the Balkan Peninsula,Especially in Greece

Zoogeography of freshwater fish fauna of the Balkan peninsula south of the Danube has been studied. Four main divisions - Ponto-Aegean, Attiko-Beotia, Dalmatia and South Adriatic-Ionian - were recognized. All endemics belong to European, Euro-Siberian or Palaearctic taxa, originated from ancestors of the Euro-Siberian stock and reached the area as well as Asia Minor in Oligocene or Miocene. Central European and Danubian species are intruders of late Pliocene to Pleistocene arriving by the freshened Black Sea and its drainage basin or directly from the North (Middle Danube) by river captures.