为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Rea...为了进一步了解和认识CuZnSOD基因的结构和功能,揭示CuZnSOD对猪抗氧化机能的影响,寻找与肉质性状相关联的分子标记,文章采用RACE(Rapid amplification of cDNA end)方法,对莱芜猪CuZnSOD基因cDNA进行克隆测序,分析其结构和功能,并用Real-time PCR检测CuZnSOD基因的表达。结果表明,CuZnSOD基因cDNA序列全长658bp(GenBank登录号:GU944822),包含76bp的5′UTR和120bp的3′UTR序列。全部CDS序列462bp,编码153个氨基酸,分子量为15.9kDa,等电点为6.03。CuZnSOD基因编码的氨基酸序列中,第3氨基酸残基处存在1个O-糖基化位点,第86氨基酸残基处存在1个N-糖基化位点。二级结构中α螺旋仅占1.31%。在进化过程中高度保守,与人、牛、小鼠和褐鼠的编码区同源性分别为87.74%、87.66%、83.44%和83.23%;氨基酸序列同源性分别为90.26%、94.12%、92.21%和91.50%。CuZnSOD存在典型的金属结合配体结构域(GFHVHQFGDNT)。基于蛋白序列所构建的分子进化树表明猪与牛的亲缘关系最近。在mRNA水平上,CuZnSOD是一个广谱表达基因,在大脑、心脏、脾脏、肝脏、肾脏、肺、大肠、小肠、脊髓、肌肉、背膘和胃中都能检测到,其在肾脏、小肠和肺中表达量较高,在心脏和肌肉组织中表达量较低。展开更多
In this paper, we have studied the interaction of CuZnSODⅢand Outer Copper, enzyme activity experiments expressed that 0.1mmol·L-1 Cu2+addition reduced the enzyme activity sharply, but this reduced action had no...In this paper, we have studied the interaction of CuZnSODⅢand Outer Copper, enzyme activity experiments expressed that 0.1mmol·L-1 Cu2+addition reduced the enzyme activity sharply, but this reduced action had not been found for the additions of 0.1mmol·L-1 1∶1 Cu2+and Zn2+, and 0.1mmol·L-1 Zn2+, respectively. This was due to the Cu2+exchanged the Zn2+in CuZnSODⅢ,and it was proved by the experiment of determination of metal content. Meanwhile, the static fluorescence quenching mechanism revealed the exist of molecular complex of CuZnSOD with Cu2+. The binding constant was obtained from lineweaver burk and double lg plot. The distance of active site to Trp is about 2.83nm, was calculated according to Frster theory.展开更多
文摘In this paper, we have studied the interaction of CuZnSODⅢand Outer Copper, enzyme activity experiments expressed that 0.1mmol·L-1 Cu2+addition reduced the enzyme activity sharply, but this reduced action had not been found for the additions of 0.1mmol·L-1 1∶1 Cu2+and Zn2+, and 0.1mmol·L-1 Zn2+, respectively. This was due to the Cu2+exchanged the Zn2+in CuZnSODⅢ,and it was proved by the experiment of determination of metal content. Meanwhile, the static fluorescence quenching mechanism revealed the exist of molecular complex of CuZnSOD with Cu2+. The binding constant was obtained from lineweaver burk and double lg plot. The distance of active site to Trp is about 2.83nm, was calculated according to Frster theory.
