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。     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特的关键作用,因而也称为微囊藻毒素去毒酶.从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列.序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920bp,其中5′-UTR长74bp,3′-UTR长174bp,编码区长672bp,编码223个氨基酸.应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878bp序列.与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用,因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp,其中5′-UTR长74 bp,3′-UTR长174 bp,编码区长672 bp,编码223个氨基酸. 应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

鲢鱼、鳙鱼、草鱼谷胱甘肽过氧化物酶cDNA的克隆及肝组织表达

利用RT-PCR技术分别从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)、鳙鱼(Aristichthys nobilis)及草食性鱼类草鱼(Ctenopharyngodon idella)肝组织扩增出谷胱甘肽过氧化物酶(GPX)cDNA核心序列。序列分析表明鲢鱼、鳙鱼、草鱼GPX的cDNA序列均为263bp,编码87个氨基酸。鲢鱼、鳙鱼、草鱼与斑马鱼(Danio rerio)(同属鲤科)、条石鲷(Oplegnathus fasciatus)、虹鳟(Oncorhynchus mykiss)(鲈形目)等鱼类GPX氨基酸序列同源性很高,为75%～93%;与人(Homo sapiens)、小鼠(Mus musculus)、大鼠(Rattus norvegicus)、猪(Sus scrofa)、牛(Bos taurus)等哺乳动物GPX氨基酸序列的同源性也较高,为76%～79%。以β-肌动蛋白为内参照,比较鲢鱼、鳙鱼、草鱼肝组织GPX cDNA的表达水平,鲢鱼、鳙鱼肝组织GPX的表达量远高于草鱼肝的GPX表达量,这与鲢鱼、鳙鱼大量摄入微囊藻毒素在鱼体内引发产生过量的脂过氧化物相适应。     

3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

鱼类谷胱甘肽S-转移酶(glutathione S-transferase,GST)是鱼类一种重要的Ⅱ相去毒酶,在催化毒素与还原谷胱甘肽(GSH)加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法,分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST(GSTpi、GSTmu、GSTtheta)基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明,鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低,可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低,可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR(RT-PCR)检测三种鱼肝脏中三型GST基因组成型表达水平,发现三种鱼各型之间皆有一定差异,尼罗罗非鱼肝脏整体GSTs基因表达很低,GSTtheta显著低于草鱼(P<0.05),GSTmu显著低于鲢鱼(P<0.05)。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。     

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。     

微囊藻毒素LR对草鱼肝脏超微结构影响的研究

关于微囊藻毒素对哺乳动物的影响已有较多的研究,指出微囊藻毒素LR是一种作用于肝脏的极强环肽毒素,并能引起肝细胞超微结构的改变［‘］,但是有关该毒素对鱼类影响的报道却很少,作者采用腹腔注射微囊藻毒素的方法,确定其在鱼体内作用的靶器官,观察超微结构的组织病理变化,并与对哺乳动物作用的结果相比较,进而探讨其对鱼类的致毒机理．回材料和方法微囊藻毒素LR由日本名城大学药学部提供。实验鱼为草鱼［Ctenopharyngodonidellus］,体重引一389,体长11—13cm,取自中国科学院水生生物研究所养殖场,在25℃下通以循环水驯化1周…     

采用套式PCR检测水库产毒微囊藻

根据所测定的微囊藻毒素合成酶mcyB基因的部分核苷酸序列,设计并筛选出两对特异性引物,用于产毒微囊藻的套式PCR检测。套式PCR针对毒素基因的检测结果与ELISA针对微囊藻毒素的检测结果相一致,但灵敏度更高。套式PCR的检测下限达1—10个微囊藻细胞/反应。采用套式PCR对广东12个主要供水水库的247份水样进行了产毒微囊藻检测,共检出阳性水样82份,阳性率为33.2%。这些阳性水样分布于除深圳水库以外的其他11个水库;其中汤溪水库水样套式PCR检出阳性率最高,达67.4%,其水样一步PCR的检出阳性率亦达25.6%,值得引起水文部门重视,并进行进一步跟踪监测。     

微囊藻毒素对鱼肝的毒性效应

研究已发现离体条件下微囊藻毒素时鱼的蛋白磷酸酶有极强的抑制作用’‘,”。有些研究证明活体致毒后细胞内蛋白的磷酸化水平增加,而毒素对生物活体致毒时蛋白磷酸酶的情况尚未见报道。本研究用鱼作为实验材料,活体致责时微囊藻毒素对鱼的蛋白磷酸酶及几种其它分子生态毒理学指标的影响。回材料与方法1．l微囊藻毒素L民根据HSYddd的方法卜‘,由日本SllWS湖水华样品中提取。l．2活体实验,对体重约259的鲫鱼腹腔注射LR,剂量分别为IO,50,100和400pg／kg。在lh和3h时取样,分别测定蛋白磷酸酶（Proteiphosphatase,简称PP）活性和…     

阴离子表面活性剂LAS对产毒和无毒微囊藻生长及产毒特性的影响

文章研究了低浓度范围内不同浓度梯度的阴离子表面活性剂直链烷基苯磺酸盐(LAS)对产毒微囊藻(Microcystis aeruginosa, FACHB905)和无毒微囊藻(Microcystis wesenbergii, FACHB908)生长、光合特性、种间竞争及毒素合成的影响。结果表明,在0.05—5.0 mg/L LAS浓度梯度处理下,产毒微囊藻的生物量、产毒基因mcyD表达量和每细胞MCs含量均在培养12d后显著增加。产毒微囊藻胞内和胞外MCs含量在各LAS浓度处理后分别为0.069、0.052、0.061、0.038和0.037 fg/fg Chl.a及107.1、103.7、127.1、99.6和113.7 ng/L。即使在0.5 mg/L低浓度LAS处理条件下,上述3个参数也分别比对照组显著增加了24.2%、12.4倍和10.4%。浓度为0—0.2 mg/L LAS对无毒微囊藻的生物量无明显影响,而较高浓度的LAS(0.5—5.0 mg/L)明显抑制了无毒微囊藻的生长。在两株微囊藻混合培养时, 0.2—1.0 mg/L LAS处理组的产毒铜绿微囊藻mcy D的表达对LAS...     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.     

Molecular characterization of glutathione peroxidase gene from the liver of silver carp, bighead carp and grass carp

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phytoplanktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.     

The activity of pro- and antioxidant systems in the liver of freshwater fishes during different seasons

The content of lipid peroxidation products--diene conjugates, lipid hydroperoxides, thiobarbituric acid reactive substances (TBARS), vitamins A, E and carotenoids and the activity of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in the liver of freshwater fishes of different species (silver carp, grass carp and common carp) in different seasons have been studied. It was established the activity of antioxidant defence system in the liver of fish depends significantly on the season and fish species. In particular, the content of lipid peroxidation products in the liver of freshwater fishes at the beginning of winter and spring was significantly higher compared to their content at the beginning of summer and autumn. The superoxide dismutase and glutathione peroxidase activities in the liver of these fish species at the beginning of winter and spring were significantly lower than at the beginning of summer and autumn while the seasonal changes of catalase activity in the liver of fish are expressed insignificantly. The content of vitamins E, A1, A2 and carotenoids in the liver of fishes of different species at the beginning of winter and spring was significantly lower than at the beginning of summer and autumn. The content of lipid peroxidation products and vitamins E, A1 and A2 in the liver of common carp is significantly lower than in the liver of silver carp and grass carp and species differences in antioxidant enzymes activity are insignificant.     

基于Miseq测序技术分析黄颡鱼不同养殖模式下池塘微生物群落结构多样性

利用Miseq测序技术分析了黄颡鱼Pelteobagrus fulvidraco (Richardson)三种养殖模式[(黄颡鱼单养、黄颡鱼混养草鱼(Ctenopharyngodon idellus, Valenciennes)和黄颡鱼混养草鱼、鳙(Aristichthys nobilis, Richardson)、鲢(Hypophthalmichthys molitrix, Valenciennes)]下养殖水体及底泥微生物群落结构多样性。结果显示, 12个样本共获得了655100条合格16S rDNA序列, 每个样本获得的有效序列数目为41256—77508, 可归纳为608—3686个分类操作单元。α多样性指数结果表明, 在三种养殖模式下末期水体的微生物群落丰度和多样性均高于养殖初期, 黄颡鱼混养草鱼模式下水体的群落丰度最高(Chao1指数: 2199.25, ACE指数: 2374.60), 黄颡鱼混养草鱼、鲢、鳙模式下水体微生物群落多样性最高(Shannon指数: 4.88, Simpson指数: 0.021)。样本聚类分析及PcoA主坐标分析结果表明, 黄颡鱼单养及黄颡鱼、草鱼混养模式下的水体和底泥的菌群组成及群落结构相似性较高, 黄颡鱼、草鱼、鲢、鳙混养模式下样本单独形成一类。从门水平的比较分析发现, 所有样本检测到细菌有8门, 其中变形菌门(Proteobacteria)的物种丰度在所有样本中占绝对优势地位。放线菌门(Actinobacteria)在养殖末期升高成为水体中占比第二的优势门, 尤其是黄颡鱼混养草鱼模式下(37.6%)。黄颡鱼在单养模式下, 养殖末期水体拟杆菌门(Bacteroidetes)细菌含量均明显增加(1.95%到10.98%)。从属水平的比较分析发现, 各样本之间的优势属组成有较大差异, 其中黄颡鱼在单养模式下水体样本中气单胞菌属(Aeromonas)在养殖末期丰度占优势(5.81%)。从微生物群落结构和多样性的角度考虑, 黄颡鱼、草鱼、鲢、鳙混养是一种值得推荐的养殖模式。     

用鲢鳙直接控制微囊藻水华的围隔试验和湖泊实践

为了控制水体富营养化所形成的水华,国外经典的生物操纵论者提倡放养食鱼性鱼类以控制食浮游生物的鱼类,藉此壮大浮游动物种群,然后借助浮游动物遏制藻类。按照这条思路,以浮游生物为食的鲢和鳙应该是清除的对象。本文作者认为我国湖泊中危害性最大的是微囊藻水华,而浮游动物根本不能摄食这种水华,不如直接利用鲢鳙进行控制,因而1989-1992年间在武汉的东湖进行了三次原位围隔试验,2000年再度重复了一次围隔试验,结果证明,迄今在东湖中已消失18年的微囊藻水华,重新出现在不养鱼的围隔里。在养入一定密度的鲢或/和鳙的围隔中,就没有水华出现。已经出现水华的围隔,放入适当密度的鲢或/和鳙后,在短期内水华又复消失;而放入草鱼的围隔,则水华始终持续,不受影响。说明鲢和鳙能有效地遏制微囊藻水华。鲢、鳙遏制水华的有效放养密度(亦即有效生物量)为46-50 g·m-3。东湖湖泊中鲢、鳙的生物量,直到1985年才达到这个水平,所以1985年湖里反常地没有出现水华。往后的年份,东湖的鱼产量越来越高,也就是湖里鲢、鳙的生物量越来越大,所以迄今没有微囊藻卷土重来的机会。东湖的水质和其他非生物条件仍适于蓝藻水华的发生与发展,如果东湖鲢、鳙的产量下降到阈值以下,亦即降到1985年以前1 000 t水平,那么微囊藻及其它蓝藻