郭慧,谭翠婷,游林玉,申玉春,卢芷程,朱春华

• 1:广东海洋大学水产学院

摘要(Abstract)：

用实时荧光定量PCR技术研究在20 mg/L亚硝酸盐胁迫下,凡纳滨对虾(Litopenaeus vannamei)肝胰腺中锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)和硫氧还蛋白(TRx)等抗氧化相关酶以及热休克蛋白HSP70、组织蛋白酶B(CTSB)的基因表达量变化。结果表明,与对照组相比,在胁迫24 h时Mn-SOD基因表达量显著升高,CAT基因表达量在12~48 h升高,GPx基因表达量在24和48 h时升高,TRx基因表达量24 h时升高,而后在48和72 h时降低,Hsp70基因表达量在48和72 h时升高,CTSB基因表达量在48 h和72 h时升高,差异均有统计学意义(P<0.05)。在亚硝酸盐胁迫下,抗氧化酶相关基因、Hsp70和CTSB基因的表达量均被显著诱导进行免疫防御,4种抗氧化相关基因和Hsp70、CTSB基因对亚硝酸盐胁迫敏感度不同,在抗亚硝酸盐胁迫过程中有一定协同作用。

关键词(KeyWords)： 亚硝酸盐;凡纳滨对虾;抗氧化基因;热休克蛋白;组织蛋白酶B;基因表达

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31600321);; 广东省自然科学基金项目(2015A030310438);; 广东海洋大学创新强校工程科研项目(GDOU2016050253);广东海洋大学优秀青年骨干教师特别资助计划(HDYQ2015003)

作者(Author): 郭慧,谭翠婷,游林玉,申玉春,卢芷程,朱春华

参考文献(References)：

• [1]汪家鑫,张钊,胡盼,等.亚硝酸氮对红鳍东方鲀的毒性效应[J].广东海洋大学学报,2013,33(6):52-56.
• [2]TACON A G J,CODY J J,CONQUEST L D,et al.Effect of culture system on the nutrition and growth performance of Pacific white shrimp Litopenaeus vannamei(Boone)fed different diets[J].Aquaculture Nutrition,2002,8(2):121-137.
• [3]FANJUL E,GRELA M A,CANEPUCCIA A,et al.The Southwest Atlantic intertidal burrowing crab Neohelice granulata modifies nutrient loads of phreatic waters entering coastal area[J].Estuarine,Coastal and Shelf Science,2008,79(2):300-306.
• [4]葛红星,李健,陈萍,等.亚硝酸盐氮胁迫下凡纳滨对虾对副溶血弧菌的易感性[J].中国水产科学,2014,21(3):629-636.
• [5]TSENG I T,CHEN J C.The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress[J].Fish&Shellfish Immunology,2004,17(4):325-333.
• [6]FURTADO P S,VALENZUELA M A J,Rodriguez-Fuentes G,et al.Chronic effect of nitrite on the rearing of the white shrimp Litopenaeus vannamei in two salinities[J].Marine and Freshwater Behaviour and Physiology,2016,49(3):201-211.
• [7]WASIELESKY W,POERSCH L H,MARTINS T G,et al.Chronic effects of nitrogenous compounds on survival and growth of juvenile pink shrimp[J].Brazilian Journal of Biology(AHEAD),2016,doi:10.1590/1519-6984.18415.
• [8]ROMANO N,ZENG C S.Subchronic exposure to nitrite,potassium and their combination on survival,growth,total haemocyte count and gill structure of juvenile blue swimmer crabs,Portunus pelagicus[J].Ecotoxicology and Environmental Safety,2009,72(4):1287-1295.
• [9]吴中华,刘昌彬,刘存仁,等.中国对虾慢性亚硝酸盐和氨中毒的组织病理学研究[J].华中师范大学学报(自然科学版),1999,33(1):119-122.
• [10]ZHENG J B,MAO Y,SU Y Q,et al.Effects of nitrite stress on m RNA expression of antioxidant enzymes,immune-related genes and apoptosis-related proteins in Marsupenaeus japonicus[J].Fish&Shellfish Immunology,2016,58:239-252.
• [11]蒋昊.中国明对虾在胁迫条件下肝胰脏的差异蛋白质组学研究[D].青岛:中国科学院研究生院(海洋研究所),2009.
• [12]郭慧,冼健安,王安利.亚硝酸盐对凡纳滨对虾血细胞毒性及p53基因表达的影响[J].水生态学杂志,2015,36(2):61-67.
• [13]GUO H,XIAN J A,LI B,et al.Gene expression of apoptosis-related genes,stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2013,157(4):366-371.
• [14]章双,黎铭,董晓慧,等.凡纳滨对虾输入蛋白α1基因的克隆与表达分析[J].广东海洋大学学报,2016,36(3):1-8.
• [15]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.
• [16]DORTS J,SILVESTRE F,TU H T,et al.Oxidative stress,protein carbonylation and heat shock proteins in the black tiger shrimp,Penaeus monodon,following exposure to endosulfan and deltamethrin[J].Environmental Toxicology and Pharmacology,2009,28(2):302-310.
• [17]Wang W N,Zhou J,Wang P,et al.Oxidative stress,DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp,Litopenaeus vannamei when exposed to acute p H stress[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2009,150(4):428-435.
• [18]LYU K,ZHU X X,CHEN R,et al.Molecular cloning of manganese superoxide dismutase gene in the cladoceran Daphnia magna:Effects of microcystin,nitrite,and cadmium on gene expression profiles[J].Aquatic Toxicology,2014,148:55-64.
• [19]汪蕾,张秀霞,郑佩华,等.硫化物胁迫对凡纳滨对虾血细胞抗氧化酶基因表达的影响[J].四川动物,2016,35(6):884-888.DOI:10.11984/j.issn.1000-7083.20160229.
• [20]张杰,洪美玲,廖广桥,等.亚硝酸盐暴露对红耳龟与中华条颈龟幼体的慢性毒性效应[J].四川动物,2011,30(2):173-177.
• [21]PLANTIVAUX A,FURLA P,ZOCCOLA D,et al.Molecular characterization of two Cu Zn-superoxide dismutases in a sea anemone[J].Free Radical Biology and Medicine,2004,37(8):1170-1181.
• [22]ZHOU J,WANG L,XIN Y,et al.Effect of temperature on antioxidant enzyme gene expression and stress protein response in white shrimp,Litopenaeus vannamei[J].Journal of Thermal Biology,2010,35(6):284-289.
• [23]GUEMOURI L,ARTUR Y,HERBETH B,et al.Biological variability of superoxide dismutase,glutathione peroxidase,and catalase in blood[J].Clinical Chemistry,1991,37(11):1932-1937.
• [24]丛艺,蒋圣娟,张士璀.硫氧还蛋白及其相关蛋白的研究进展[J].海洋科学,2008,32(6):79-84.
• [25]刘雷,尹钧.硫氧还蛋白的研究[J].东北农业大学学报,2003,34(2):219-225.
• [26]HUANG W J,LEU J H,TSAU MT,et al.Differential expression of Lv HSP60 in shrimp in response to environmental stress[J].Fish&Shellfish Immunology,2011,30(2):576-582.
• [27]Rungrassamee W,Leelatanawit R,Jiravanichpaisal P,et al.Expression and distribution of three heat shock protein genes under heat shock stress and under exposure to Vibrio harveyi in Penaeus monodon[J].Developmental&Comparative Immunology,2010,34(10):1082-1089.
• [28]QIAN Z Y,LIU X L,WANG L J,et al.Gene expression profiles of four heat shock proteins in response to different acute stresses in shrimp,Litopenaeus vannamei[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2012,156(3/4):211-220.
• [29]TURK B,STOKA V.Protease signalling in cell death:caspases versus cysteine cathepsins[J].FEBS Letters,2007,581(15):2761-2767.
• [30]ZHENG L,K?GEDAL K,DEHVARI N,et al.Oxidative stress induces macroautophagy of amyloidβ-protein and ensuing apoptosis[J].Free Radical Biology and Medicine,2009,46(3):422-429.
• [31]ZHANG Z B,LI Z G.Cathepsin B and phospo-JNK in relation to ongoing apoptosis after transient focal cerebral ischemia in the rat[J].Neurochemical Research,2012,37(5):948-957.
• [32]GRIFFITT R J,GREIG T W,CHANDLER G T,et al.Serial analysis of gene expression reveals identifiable patterns in transcriptome profiles of Palaemonetes pugio exposed to three common environmental stressors[J].Environmental Toxicology and Chemistry,2007,26(11):2413-2419.
• [33]WANG X M,LIU B Z,WANG G D,et al.Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix[J].Aquaculture,2008,282(1/4):41-46.
• [34]STEPHENS A,ROJO L,ARAUJO-BERNAL S,et al.Cathepsin B from the white shrimp Litopenaeus vannamei:c DNA sequence analysis,tissues-specific expression and biological activity[J].Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2012,161(1):32-40.