斧文蛤金属硫蛋白基因的克隆与表达分析
CLONING AND EXPRESSION OF METALLOTHIONEIN GENE IN MERETRIX LAMARCKII
-
摘要: 金属硫蛋白（Metallothionein，MT）是一类富含半胱氨酸的小分子蛋白质，参与机体重金属解毒、维持金属元素代谢平衡以及清除自由基等生理功能。为了解斧文蛤金属硫蛋白（Ml-MT）的分子生物学特征及其在重金属Cd2+胁迫下的响应机制，本文采用RACE技术从斧文蛤（Meretrix lamarckii）总RNA反转录产物中获得了636 bp的Ml-MT cDNA基因序列。该序列包含65 bp的5非编码区（UTR）和340 bp的3非编码区（UTR）以及231 bp的开放阅读框（ORF），可编码76个氨基酸；其中半胱氨酸占27%，不含芳香族氨基酸，含16个MT所特有的Cys-Xn-Cys结构，预测的分子量约为7.704 kD，理论等电点7.138。MT氨基酸序列比对分析表明：斧文蛤金属硫蛋白（Ml-MT）与丽文蛤（Meretrix lusoria）的相似性高达88%，与文蛤（Meretrix meretrix）的同源性为87%。实时荧光定量（qRT-PCR）检测MT在斧文蛤5种组织中均有表达，但存在组织特异性，其中内脏团表达量最高，其次依次为鳃丝、闭壳肌、外套膜、斧足。在Cd2+（0.13 mg/L）胁迫0、6h、12h、24h、48h、72h和96h下，斧文蛤内脏团MT呈现出不同程度的上调表达，具体表现为高-低-高-低的波浪式变化，除6h以外，其他时间点均与对照组存在极显著差异（P0.01）。本研究表明：MT基因在维护机体正常生理功能及斧文蛤抵御重金属Cd2+胁迫过程中发挥着重要的分子调控作用。Abstract: Metallothionein (MT) is a family of cysteine-rich, low molecular weight proteins. It involves heavy metal detoxification, maintain metabolic balance free radical scavenging metal elements and other physiological functions. A large number of studies show that the expression level of MT can indirectly reflect the stress response of organisms to the environment and the ability of detoxification of heavy metals. As a result, it is often used to evaluate the pollution status of heavy metals and the adaptability of organisms to these heavy metals. Therefore, MT presently has been selected as one of the biomarkers of marine environmental monitoring by the United Nations Environment Program. In this study, by using RACE technology to gain 636 bp of metallothionein (Ml-MT) gene cDNA sequences from total RNA reverse transcription product. The sequence contains 65 bp of 5' Untranslated Region (UTR) and 340 bp of 3' UTR in addition to 213 bp of the open reading frame (ORF), which can encode 76 amino acids. Of this, accounting for 27% cysteine, no aromatic amino acids and there are 16 MT of the specific Cys-Xn-Cys structure, where the molecular weight and theoretical isolectric point were predicted to be 7.704 kD and 7.138 respectively. The MT amino acid sequence alignment comparative analysis indicated that the M. lamarckii and Meterix lusoria were similar at more than 88%, with M. meretrix homology of 87%. The quantification real-time PCR (qRT-PCR) detection showed that there were MT expressions in five kinds of tissue of M. meretrix. These expressions had tissue specificity where visceral mass had the highest levels, followed by the Gill filaments, adductor muscle, mantle and foot. In Cd2+ 0.13 mg/L exposures of 0, 6h, 12h, 24h, 48h, 72h and 96h, the MT mRNA of M. meretrix visceral mass displayed different increasing degrees of contamination time. Specific performance is high-low-high-low wave trend changes. Except for 6h, the other time points compare with control group have significant difference (P0.01). This study showed that MT gene in the maintenance of normal physiological function of the body play an important role of molecular regulation in Cd2+ stress of M. meretrix.
-
Keywords:
- Meretrix lamarckii /
- Metallothionein /
- Gene cloning /
- Expression /
- Cd2+
-
-
[1] Zhuang Q Q. Chinese Animal Fauna:Mollusca Bivalvia Veneridae[M]. Beijing:Science Press. 2001, 229-236[庄启谦. 中国动物志-软体动物门双壳纲帘蛤科. 北京:科学出版社. 2001, 229-236]
[2] Wang X Q, Cao M, Yan B L, et al. Biology and reproduction of clam Cyclinasinensis[J]. Fisheries Science, 2006, 25(6):312-316[王兴强, 曹梅, 阎斌伦, 等. 青蛤的生物学及其繁殖. 水产科学, 2006, 25(6):312-316]
[3] Pan B P, Song X, Luo K Y, et al. expression of lysozyme gene in Cyclinasinensis exposed to Vibrio anguillarum[J]. Oceanologia et Limnologia Sinica, 2010, 41(6):901-906[潘宝平, 宋欣, 罗凯娅, 等. 青蛤(Cyclinasi-nensis)溶菌酶基因在鳗弧菌(Vibrio anguillarum)刺激下的表达. 海洋与湖沼, 2010, 41(6):901-906]
[4] Dziegiel P, Jelena M, Muszczynska B, et al. Role of metallothionein expression in non-small cell Lung carcinomas[J]. RocznikiAkademiiMedycznej W Bialymstoka, 2004, 49(1):43-45
[5] Liu W Q, Niu D J, Song L S, et al. Cloning and expression of Metallothionein gene in Argopectenirradians[J]. Oceanologia et Limnologia Sinica, 2006, 37(5):444-449[刘维青, 倪多娇, 宋林生, 等. 海湾扇贝(Argopectenirradians)金属硫蛋白基因的隆与分析. 海洋与湖沼, 2006, 37(5):444-449]
[6] Zhang G C. Functions and applied prospects of metallothionein[J]. Yantai Normal University Journal (Natural Science Edition), 2005, 21(1):142-145[张桂春. 金属硫蛋白的功能及应用前景. 烟台师范学院学报(自然科学版), 2005, 21(1):142-145]
[7] UNEP/RAMOGE. Manual on the biomarkers recommended for the MED POL biomonitoring programme[R]. UNEP, Athens. 1999, 1-92
[8] Ridlington J W, Fowler B A. Isolation and partial characterization of a cadmium-binding protein from the Ameri-can oyster (Crassostrea virginica)[J]. Chemico-Biologi-cal Interactions, 1979, 25(2/3):127-138
[9] Lemoine S, Laulier M. Potential use of the levels of the mRNA of a specific metallothionein isoform (MT220) in mussel (Mytilus edulis) as a biomarker of cadmium contamination[J]. Marine Pollution Bulletin, 2003, 46(11):1450-1455
[10] Wang L L, Song L S, Ni D J, et al. Alteration of metallothionein mRNA in bay scallop Argopectenirradians under cadmium exposure and bacteria challenge[J]. Comparative Biochemistry and Physiology, Part C, 2009, 149(1):50-57
[11] Lu D, Luo K Y, Pan B P, et al. Cloning and expression of metallothionein and thioredoxin gene in Cyclina sinensis exposed to cadmium[J]. Oceanologia et Limnologia Si-nica, 2012, 43(1):47-51[吕达, 罗凯娅, 潘宝平, 等. 青蛤(Cyclina sinensis)金属硫蛋白及硫氧还蛋白基因的克隆与表达分析. 海洋与湖沼, 2012, 43(1):47-51]
[12] Lin X L, Wang Q, Yuan Z Y, et al. Cloning and expression of Metallothionein gene in Ruditapes philippinarum exposed to cadmium[J]. Oceanologia et Limnologia Si-nica, 2014, 45(5):1006-1012[林晓玲, 王清, 袁泽轶, 等. 菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白基因克隆及Cd2+胁迫下的表达分析. 海洋与湖沼, 2014, 45(5):1006-1012]
[13] Jiang G P, Xiao G Q, Zhang J M, et al. Effect of ocean acidification on the acute toxicity of Cd2+ and Hg2+ to juvenile Meretrix lamarckii[J]. Acta Hydrobiologica Si-nica, 2016, 40(2):1-7[蒋国萍, 肖国强, 张炯明, 等. 海洋酸化下Cd2+和Hg2+对斧文蛤幼贝急性毒性效应. 水生生物学报, 2016, 40(2):1-7]
[14] Wang L, Pan L Q, Xu C Q, et al. Combination effects of cadmium and benzo[]pyrene on acute toxicity and metabolic enzymes activities of Ruditapes philippinarum[J]. Acta Hydrobiologica Sinica, 2011, 35(1):37-44[王琳, 潘鲁青, 徐超群, 等. Cd~(2+)-B[]P复合污染对菲律宾蛤仔急性毒性和解毒代谢酶活力的影响. 水生生物学报, 2011, 35(1):37-44]
[15] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-Ct method[J]. Methods, 2001, 25(4):402-408
[16] Vasak M, Hasler D W. Metallothionein:new functional and structural insights[J]. Current Opinion in Chemical Biology, 2000, 4(2):177-183
[17] Coyle P, Philcox J C. Metallothionein:The multipurpose protein[J]. Cellular and Molecular Life Sciences, 2002, 59(4):627-647
[18] Ruttkay-Nedecky B, Nejdl L, Gumulec J, et al. The role of metallothionein in oxidative stress[J]. International Journal of Molecular Sciences, 2013, 14(3):6044-6066
[19] Ren X Y, Zhou Y, Zhang J P, et al. Metallothionein gene expression under different time in testicular sertoli and spermato genic cells of rats treated with cadmium[J]. Reproductive Toxicology, 2003, 17(2):219-227
[20] Moulis J M. Cellular mechanisms of cadmium toxicity related to the homeostasis of essential metals[J]. Biometals, 2010, 23(5):877-896
[21] Choi Y K, Jo P G, Choi C Y. Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas[J]. Comparative Biochemistry and Physiology, Part C:Toxicology and Pharmacology, 2008, 147(3):286-292
[22] Zhao S S, Meng F P, Fu H F, et al. Metallothionein levels in gills and visceral mass of Ruditapes philippinarum exposed to sublethal doses of cadmium and copper[C]. International Conference on Challenges in Environmental Science and Computer Engineering, CESCE. 2010, 2:181-186
[23] Wu C, Chen L X, Ni X Y, et al. Metallothionein in diffe-rent tissues of meretrixmeretrix exposed to Zinc[J]. Sichuan Journal of Zoology, 2012, 31(1):54-58[吴超, 陈露茜, 倪小英, 等. 锌诱导金属硫蛋白在文蛤不同组织中的表达. 四川动物, 2012, 31(1):54-58]
[24] Cao J H, Ma G Z, Fang Z Q. Effects of cadmium on superoxide dismutase activities in the liver and gill tissue of grass carp[J]. Reservoir Fisheries, 2004, 24(1):9-11
[25] Amiard-Triquet C, Rainglet F, Larroux C, et al. Metallothioneins in Arctic bivalves[J]. Ecotoxicology and Envi-ronmental Safety, 1998, 41(1):96-102
[26] Choi Y K, Jo P G, Choi C Y. Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas[J]. Comparative and Physiology, Part C, 2008, 147(3):286-292
[27] Chang Y T, Jong K J, Liao B K, et al. Cloning and expression of metallothionein cDNA in the hard clam (Meretrix lusoria) upon cadmium exposure[J]. Aquaculture, 2007, 262(2-4):504-513
[28] Wang Q, Wang X, Wang X, et al. Analysis of metallotione in expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium expo-sure[J]. Aquatic Toxicology, 2010, 100(4):321-328
[29] Paul-Pont I, Gonzalez P, Baudrimont M, et al. Short-term metallothionein inductions in the edible cockle Cerastoderma edule after cadmium or mercury exposure:Discre-pancy between mRNA and protein responses[J]. Aquatic Toxicology, 2010, 97(3):260-267
[30] Huo L H, Chen C F, Lin Z H, et al. The metallothionein gene expression in Sinonovacula constricta exposing to cadmium[J]. Oceanologia et Limnologia Sinica, 2012, 43(4):723-728[霍礼辉, 陈彩芳, 林志华, 等. 镉诱导缢蛏(Sinonovacula constricta)体内金属硫蛋白基因变化规律研究. 海洋与湖沼, 2012, 43(4):723-728]
[31] Baer K N, Benson W H. Influence of chemical and envi-ronmental stressors on acute cadmium toxicity[J]. Jour-nal of Toxicology Environmental Health, 1987, 22(1):35-44
-
期刊类型引用(5)
1. 陈琛, 蔺蓓蓓, 徐尤美, 李鑫鑫, 刘祥. 文蛤生物活性多肽/蛋白研究进展. 海洋渔业. 2019(03): 374-384 . 
百度学术
2. 强承魁, 秦越华, 张明, 梁玉莹, 曹丹, 丁永辉. 小麦金属硫蛋白基因的克隆与组织表达分析. 分子植物育种. 2019(14): 4518-4523 . 百度学术
3. 陈涛, 陈逸平, 黄鑫, 彭婷婷, 王晓静, 唐玲, 黄献培. 海水中重金属生物监测分子指示物研究进展. 环境科学与技术. 2019(08): 86-94 . 百度学术
4. 朱晓莹, 孟霄, 姚海洋, 许星鸿. 海洋生物金属硫蛋白开发利用研究进展. 湖北农业科学. 2018(10): 9-13 . 百度学术
5. 谢清清, 陈彩芳, 吴林德, 林志华. 铅(Pb)胁迫对泥蚶血液MT及Vg基因表达的诱导效应研究. 海洋科学. 2017(11): 75-81 . 百度学术
其他类型引用(1)
计量
- 文章访问数: 1358
- HTML全文浏览量: 1
- PDF下载量: 102
- 被引次数: 6
下载: