真鲷去毒相关基因cDNA序列的克隆与肝脏组成型表达分析

Molecular Cloning of Detoxification-Related Genes and Liver Constitutive Expression in Red Sea Bream Pagrus major

赤潮发生时产生的一些海洋生物毒素对人类和海洋动物的安全形成潜在的威胁甚至导致死亡.为从分子水平探讨鱼类中海洋藻毒素的去毒分子机理,采用RT-PCR法克隆了真鲷(Pagrus major)肝脏芳香烃受体核转位蛋白(ARNT)和I时相异生素代谢酶细胞色素P4501A(CYP1A)基因cDNA核心序列,同时,应用半定量RT-PCR方法,以β-肌动蛋白作为外参照,在其指数期增长的范围内研究了芳香烃受体(AHR)、ARNT、CYP1A、II时相异生素代谢酶alpha型谷胱甘肽S-转移酶(GSTA1、GSTA2)、rho型谷胱甘肽S-转移酶(GSTR)、热休克蛋白70(HSP70)基因组成型表达水平.结果发现,真鲷ARNT、CYP1A基因cDNA核心序列片段分别长438bp和908bp,分别编码146和302个氨基酸.序列同源性分析发现,真鲷与门齿鲷(Stenotomus chrysops)、石首鱼(Micropogon undulatus)ARNT基因氨基酸序列同源性高达97.2%、95.2%,与斑马鱼(Brachydanio rerio)、人、大鼠、小鼠ARNT同源性较低(77.2%～79.3%).真鲷与门齿鲷、金头鲷(Sparus auratus)、欧洲川鲽(Rhombus maximus)、欧洲海鲈(Dicentrachus labrax)CYP1A基因氨基酸序列同源性较高,为84.8%～94.0%,与斑马鱼、人、小鼠CYP1A同源性较低,为59.6%～77.8%.真鲷肝脏AHR、ARNT、CYP1A、GSTA1、GSTA2、GSTR和HSP70基因组成型表达水平分别为(25.32±6.56)%、(26.22±4.24)%、(146.5±16.06)%、(55.42±3.75)%、(48.82±10.89)%、(79.47±3.13)%、(107.42±14.34)%.

Partial cDNA sequences of hepatic aryl hydrocarbon receptor nuclear translocator（ARNT）and cytochrome P-450 1A （CYP1A）genes from red sea bream （Pagrus major）were obtained by PCR using degenerate primers. The constitutive transcriptional expression of the aryl hydrocarbon receptor（AHR）, ARNT, CYPIA, phase Ⅱ（glutathione S-transferase alpha 1, GSTA1; glutathione S-transferase alpha 2, GSTA2; glutathione S-transferase rho, GSTR）xenobiotic metabolizing enzymes and heat shock protein 70（ HSP70 ）were also investigated. Results showed that, the cDNA sequences of ARNT and CYPIA genes were 438bp and 908bp in length, encoding 146 and 302 amino acids, respectively. Homology of the ARNT amino acid sequence was high （97.2%, 95.2% ）with scup （Stenotomus chrysops ）and Atlantic croaker （Micropogon undulatus ） ARNT, and was modest （77.2%-79.3%）with zebrafish （Brachydanio rerio）, human, rat and mouse ARNT. The CYPIA amino acid sequence showed a high conservation with scup, gilthead seabream （Sparus auratus）, European flounder （Rhombus maximus ）, European seabass （Dicentrachus labrax）（ 84.8%-94.0% ）and a low conservation with zebrafish, human and mouse（59.6%-77.8%）. The AHR, ARNT, CYP1A, GSTA1, GSTA2, GSTR and HSP70 mRNA abundance in red sea bream were （ 25.32 ±6.56 ）%, （ 26.22 ±4.24 ）%, （ 146.5 ± 16.06 ）%, （ 55.42 ±3.75 ）%, （48.82±10.89 ）%, （ 79.47±3.13 ） % and （ 107.42±14.34 ） %, respectively.