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

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Cd^(2+)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 c DNA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 k D,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(q RT-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Cd^(2+)(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(P<0.01)。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Cd^(2+)胁迫过程中发挥着重要的分子调控作用。

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 （P〈0.01）. 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.