微囊藻毒素-LR对鱼类分子毒性效应的研究
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摘要
随着水体富营养化加剧，藻类所引起的水污染问题已越来越引起人们的关注，蓝藻是目前已知产生毒素最多、对人类健康造成的危害最大的藻类。微囊藻毒素-LR(microcystins-LR，MC-LR)是有毒蓝藻细胞合成的二级代谢产物，是一种细胞内毒素。微囊藻毒素可影响鱼类的胚胎发育和生长，也可引起肝脏、肾脏等内部器官的病理变化，和导致鱼体的氧化损伤等。然而，有关微囊藻毒素对鱼类分子水平上的毒性效应的研究则很少。本研究以斑马鱼和鳙鱼为对象，采用实时荧光定量PCR、分子克隆和免疫印迹技术在分子水平上揭示了MC-LR对鱼类的毒理效应。
本研究中采用人工繁殖的方法收集健康的斑马鱼胚胎，待孵化出膜后，用浓度为200μg／L和800μg／L MC-LR溶液浸泡幼鱼进行攻毒实验。在染毒12h，24h，48h，96h和168h后取样。用实时荧光定量PCR方法检测了斑马鱼幼鱼的重组激活基因Rag(Rag1，Rag2)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、T细胞受体α(TCRα)、转录因子GATA1、锌指纹蛋白(Ikaros)和热休克蛋白基因(HSP90、HSP70、HSP60、HSP27)的表达变化。发现Rag1、Rag2、LCK、TCRα、GATA1和Ikaros都有表达上升趋势，特别是在800μg／L MC-LR处理后，在很多时间点内表达上升明显，与对照组有显著差异(P＜0.05)；热休克蛋白基因(HSP90、HSP70、HSP60、HSP27)的表达变化更为明显，在200μg／L和800μg／L MC-LR处理后，所有热休克蛋白基因的表达均有上升，且差异明显(P＜0.05)，尤其是HSP70在800μg／L MC-LR处理168h后，其表达量上升了近300倍。这说明MC-LR可影响斑马鱼幼鱼早期发育的重要调控因子、转录因子和热休克蛋白的表达，从而推测MC-LR可能影响斑马鱼的早期发育和免疫系统功能。
本研究通过分子克隆技术中的RACE方法获得了两个鳙鱼去毒酶相关基因的cDNA全长，并进行了系统进化分析及推导的蛋白质序列分析。
谷胱甘肽过氧化物酶(Glutathione Peroxidase，GPX)基因cDNA全长903个核苷酸(nucleotides，nt)(GenBank accession no．FJ357422)，包含426 nt的开放阅读框，编码142个氨基酸，其中5′、3′端非编码区分别为182 nt和292 nt，推测GPX蛋白的分子式C_(740)H_(1144)N_(196)O_(213)S_4，分子量为16.3226千道尔顿，等电点5.93，预测该蛋白为水溶性。系统进化分析和氨基酸序列比对的结果表明，鳙鱼GPX与草鱼、鲢鱼、鲋鱼的相似性较高，分别为99.3％，97.9％，93.0％。
谷胱甘肽还原酶(Glutathione reductase，GR)基因cDNA全长831个核苷酸(nucleotides，nt)(GenBank accession no．FJ349627)，包含717 nt的开放阅读框，编码239个氨基酸，其5′、3′端非编码区分别为74 nt和40 nt，推测GR蛋白的分子式C_(1131)H_(1796)N_(324)O_(347)S_7，分子量为25.709千道尔顿，等电点7.71，预测该蛋白为水溶性。系统进化分析和氨基酸序列比对的结果表明，鳙鱼GR与斑马鱼、大西洋鲑相似性较高，分别为90.8％和81.1％。
在健康鳙鱼中，对两个基因转录和表达产物的器官组织分布进行了详细的分析。实时荧光定量PCR的结果表明，GPX和GR在所有被检测的鳙鱼组织中都有表达，呈组成型表达，且具有相似的组织分布模式，即在肝脏中的表达最高，其次是脾脏、鳃、中肾、头肾等，脑和心脏中的表达较低。分别构建GPX和GR基因的原核表达质粒pQE40-GPX和pQE40-GR，通过原核表达、表达蛋白纯化、并制备多克隆抗体，对脑、鳃、心脏、头肾、中肾、肝脏、肌肉、脾脏、和胸腺等器官中的目的基因蛋白进行免疫印迹分析。免疫印迹Western blotting结果显示GPX蛋白在各个组织中广泛表达。
经腹腔注射MC-LR(50μg／kg body weight)对鳙鱼进行染毒实验，以注射灭菌生理盐水作为对照，分别于染毒后6h，12h，24h，48h，96h和168h后取处理组和对照组的肝脏、脾脏、头肾、肠道。用实时荧光定量PCR方法检测鳙鱼的GPX和GR表达变化。发现在MC-LR诱导后GPX在肝脏、脾脏和头。肾中过量表达，差异显著(P＜0.05)；GR在肠道和头肾中，表达变化显著(P＜0.05)，上升倍数较高，而在肝脏和脾脏中GR的表达虽然也是增加的，但变化没有肠道和头肾中的明显。结果表明，GPX和GR基因在微囊藻毒素的影响下，转录及表达加强，生成更多的酶蛋白，从而增强机体对微囊藻毒素的解毒能力。
Microcystins are cyanobacterial toxins in water blooms that have received increasingattention as a public biohazard for human and animal health. Microcystins are a group ofcyanobacterial hepatotoxins and the most common and potently toxic one ismicrocystin-LR (MC-LR). It has been well documented that MC-LR may have adverseeffect on embryo development and growth of fish, and pathological damages in liver andkidney of fish, and oxidative damage in fish exposed to MC-LR. However, the moleculartoxicology of MC-LR on fish has not yet been investigated. The present study wastherefore designed to examine the molecular toxic effect of MC-LR on bighead carpAristichthys nobilis and zebrafish Danio rerio by using real-time polymerase chainreaction, molecular cloning and immunoblotting.
Zebrafish embryos were collected from artificial fertilization. The transcriptions ofsix immune-related genes, i.e. Rag1, Rag2, Ikaros, TCRα, Lck, GATA1 and HSPs(HSP90、HSP70、HSP60、HSP27) were analyzed by real-time PCR at 12h, 24h, 48h,96h and 168h post immersing in 200μg/L and 800μg/L MC-LR, respectively. The increasein transcriptions of Ragl, Rag2, Ikaros, TCRα, Lck and GATA1 were observed, andsignificant higher transcriptions of those six genes were observed on fish exposed to800μg/L MC-LR. There were significant increases in transcriptions of HSPs, andespecially significant change on HSP70 of fish exposed to 800μg/L MC-LR at 168h.These results demonstrated that microcystin-LR exposure could affect the function ofzebrafish larval immune system, resulting in immunomodulation.
The GPX and GR cDNA full sequence of Aristichthys nobilis has been cloned usingRACE-PCR and PCR.
The GPX full length cDNA is 903 bp, including a 182 bp 5'UTR (untranslatedregion),a 426 bp open reading frame,and a 292 bp 3'UTR. The putative protein of GPX is142 aa. The molecular formula is C_(740)H_(1144)N_(196)O_(213)S_4, and the molecular weight is16.3226KDa, and estimated pIs is 5.93. GPX protein is water solubility.Comparison of thededuced amnio acid sequences showed that GPX of Aristichthys nobilis shared 99.3%,97.9% and 93.0% identity with those of Ctenopharyngodon idella, Hypophthalmichthysmolitrix and Carassius auratus, respectively.
The GR full length cDNA is 831 bp, including a 74 bp 5'UTR (untranslated region),a 717 bp open reading frame, and a 40 bp 3'UTR. The putative protein of GR is 239 aa. The molecular formula is C_(1131)H_(1796)N_(324)O_(347)S_7, and the molecular weight is 25.709KDa,and estimated pls is 7.71. GRprotein is water solubility. Comparison of the deduced amnioacid sequences showed that GR of Aristichthys nobilis shared 90.8% and 81.1%identitywith those of Danio rerio and Salmo salar, respectively.
The organ distribution of GPX and GR was analyzed both in the transcription andprotein levels.By real time PCR analysis, the transcription products of the cloned geneswere detected in different tissues of healthy bighead carp, and the two genes had similiarpatterns of tissue distribution, with the highest expression level in liver. Western blottinganalyses revealed that GPX protein existed broadly in examined tissues
The gene transcription of GPX and GR in liver, spleen, head kidney and intestine ofAristichthys nobilis were quantified by real-time PCR at 6h, 12h, 24h, 48h, 96h and 168hpost intraperitoneal injection with 50μg MC-LR/kg body weight. Significant increase ofGPX in liver, spleen and head kidney were observed. Also, significant increases of GR inintestine and head kidney were observed, and there were no significant changes of GR inliver and spleen. These results showed that the increased transcriptions of GPX and GR inAristichthys nobilis could be induced by MC-LR, leading to generating more antioxidaseand elevating the ability of detoxification on fish.
本研究中采用人工繁殖的方法收集健康的斑马鱼胚胎，待孵化出膜后，用浓度为200μg／L和800μg／L MC-LR溶液浸泡幼鱼进行攻毒实验。在染毒12h，24h，48h，96h和168h后取样。用实时荧光定量PCR方法检测了斑马鱼幼鱼的重组激活基因Rag(Rag1，Rag2)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、T细胞受体α(TCRα)、转录因子GATA1、锌指纹蛋白(Ikaros)和热休克蛋白基因(HSP90、HSP70、HSP60、HSP27)的表达变化。发现Rag1、Rag2、LCK、TCRα、GATA1和Ikaros都有表达上升趋势，特别是在800μg／L MC-LR处理后，在很多时间点内表达上升明显，与对照组有显著差异(P＜0.05)；热休克蛋白基因(HSP90、HSP70、HSP60、HSP27)的表达变化更为明显，在200μg／L和800μg／L MC-LR处理后，所有热休克蛋白基因的表达均有上升，且差异明显(P＜0.05)，尤其是HSP70在800μg／L MC-LR处理168h后，其表达量上升了近300倍。这说明MC-LR可影响斑马鱼幼鱼早期发育的重要调控因子、转录因子和热休克蛋白的表达，从而推测MC-LR可能影响斑马鱼的早期发育和免疫系统功能。
本研究通过分子克隆技术中的RACE方法获得了两个鳙鱼去毒酶相关基因的cDNA全长，并进行了系统进化分析及推导的蛋白质序列分析。
谷胱甘肽过氧化物酶(Glutathione Peroxidase，GPX)基因cDNA全长903个核苷酸(nucleotides，nt)(GenBank accession no．FJ357422)，包含426 nt的开放阅读框，编码142个氨基酸，其中5′、3′端非编码区分别为182 nt和292 nt，推测GPX蛋白的分子式C_(740)H_(1144)N_(196)O_(213)S_4，分子量为16.3226千道尔顿，等电点5.93，预测该蛋白为水溶性。系统进化分析和氨基酸序列比对的结果表明，鳙鱼GPX与草鱼、鲢鱼、鲋鱼的相似性较高，分别为99.3％，97.9％，93.0％。
谷胱甘肽还原酶(Glutathione reductase，GR)基因cDNA全长831个核苷酸(nucleotides，nt)(GenBank accession no．FJ349627)，包含717 nt的开放阅读框，编码239个氨基酸，其5′、3′端非编码区分别为74 nt和40 nt，推测GR蛋白的分子式C_(1131)H_(1796)N_(324)O_(347)S_7，分子量为25.709千道尔顿，等电点7.71，预测该蛋白为水溶性。系统进化分析和氨基酸序列比对的结果表明，鳙鱼GR与斑马鱼、大西洋鲑相似性较高，分别为90.8％和81.1％。
在健康鳙鱼中，对两个基因转录和表达产物的器官组织分布进行了详细的分析。实时荧光定量PCR的结果表明，GPX和GR在所有被检测的鳙鱼组织中都有表达，呈组成型表达，且具有相似的组织分布模式，即在肝脏中的表达最高，其次是脾脏、鳃、中肾、头肾等，脑和心脏中的表达较低。分别构建GPX和GR基因的原核表达质粒pQE40-GPX和pQE40-GR，通过原核表达、表达蛋白纯化、并制备多克隆抗体，对脑、鳃、心脏、头肾、中肾、肝脏、肌肉、脾脏、和胸腺等器官中的目的基因蛋白进行免疫印迹分析。免疫印迹Western blotting结果显示GPX蛋白在各个组织中广泛表达。
经腹腔注射MC-LR(50μg／kg body weight)对鳙鱼进行染毒实验，以注射灭菌生理盐水作为对照，分别于染毒后6h，12h，24h，48h，96h和168h后取处理组和对照组的肝脏、脾脏、头肾、肠道。用实时荧光定量PCR方法检测鳙鱼的GPX和GR表达变化。发现在MC-LR诱导后GPX在肝脏、脾脏和头。肾中过量表达，差异显著(P＜0.05)；GR在肠道和头肾中，表达变化显著(P＜0.05)，上升倍数较高，而在肝脏和脾脏中GR的表达虽然也是增加的，但变化没有肠道和头肾中的明显。结果表明，GPX和GR基因在微囊藻毒素的影响下，转录及表达加强，生成更多的酶蛋白，从而增强机体对微囊藻毒素的解毒能力。
Microcystins are cyanobacterial toxins in water blooms that have received increasingattention as a public biohazard for human and animal health. Microcystins are a group ofcyanobacterial hepatotoxins and the most common and potently toxic one ismicrocystin-LR (MC-LR). It has been well documented that MC-LR may have adverseeffect on embryo development and growth of fish, and pathological damages in liver andkidney of fish, and oxidative damage in fish exposed to MC-LR. However, the moleculartoxicology of MC-LR on fish has not yet been investigated. The present study wastherefore designed to examine the molecular toxic effect of MC-LR on bighead carpAristichthys nobilis and zebrafish Danio rerio by using real-time polymerase chainreaction, molecular cloning and immunoblotting.
Zebrafish embryos were collected from artificial fertilization. The transcriptions ofsix immune-related genes, i.e. Rag1, Rag2, Ikaros, TCRα, Lck, GATA1 and HSPs(HSP90、HSP70、HSP60、HSP27) were analyzed by real-time PCR at 12h, 24h, 48h,96h and 168h post immersing in 200μg/L and 800μg/L MC-LR, respectively. The increasein transcriptions of Ragl, Rag2, Ikaros, TCRα, Lck and GATA1 were observed, andsignificant higher transcriptions of those six genes were observed on fish exposed to800μg/L MC-LR. There were significant increases in transcriptions of HSPs, andespecially significant change on HSP70 of fish exposed to 800μg/L MC-LR at 168h.These results demonstrated that microcystin-LR exposure could affect the function ofzebrafish larval immune system, resulting in immunomodulation.
The GPX and GR cDNA full sequence of Aristichthys nobilis has been cloned usingRACE-PCR and PCR.
The GPX full length cDNA is 903 bp, including a 182 bp 5'UTR (untranslatedregion),a 426 bp open reading frame,and a 292 bp 3'UTR. The putative protein of GPX is142 aa. The molecular formula is C_(740)H_(1144)N_(196)O_(213)S_4, and the molecular weight is16.3226KDa, and estimated pIs is 5.93. GPX protein is water solubility.Comparison of thededuced amnio acid sequences showed that GPX of Aristichthys nobilis shared 99.3%,97.9% and 93.0% identity with those of Ctenopharyngodon idella, Hypophthalmichthysmolitrix and Carassius auratus, respectively.
The GR full length cDNA is 831 bp, including a 74 bp 5'UTR (untranslated region),a 717 bp open reading frame, and a 40 bp 3'UTR. The putative protein of GR is 239 aa. The molecular formula is C_(1131)H_(1796)N_(324)O_(347)S_7, and the molecular weight is 25.709KDa,and estimated pls is 7.71. GRprotein is water solubility. Comparison of the deduced amnioacid sequences showed that GR of Aristichthys nobilis shared 90.8% and 81.1%identitywith those of Danio rerio and Salmo salar, respectively.
The organ distribution of GPX and GR was analyzed both in the transcription andprotein levels.By real time PCR analysis, the transcription products of the cloned geneswere detected in different tissues of healthy bighead carp, and the two genes had similiarpatterns of tissue distribution, with the highest expression level in liver. Western blottinganalyses revealed that GPX protein existed broadly in examined tissues
The gene transcription of GPX and GR in liver, spleen, head kidney and intestine ofAristichthys nobilis were quantified by real-time PCR at 6h, 12h, 24h, 48h, 96h and 168hpost intraperitoneal injection with 50μg MC-LR/kg body weight. Significant increase ofGPX in liver, spleen and head kidney were observed. Also, significant increases of GR inintestine and head kidney were observed, and there were no significant changes of GR inliver and spleen. These results showed that the increased transcriptions of GPX and GR inAristichthys nobilis could be induced by MC-LR, leading to generating more antioxidaseand elevating the ability of detoxification on fish.
引文
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