坛紫菜hsp70基因真核表达载体的构建与转化

坛紫菜是极具经济效益的大型海藻,生活在环境多变的潮间带,易受到温度、渗遗压和辐射等因素剧烈变化的影响,经常处于逆境胁迫中.热休克蛋白70(HSP70)是生物体内一种重要且高度保守的应激因子,作为分子伴侣在胁迫条件下首先被诱导出来,在逆境胁迫调节中起着重要的作用.构建紫菜hsp70基因真核表达体系对于了解该基因在紫菜抗逆过程中的作用有重要意义.利用PCR技术扩增得到大小为1.89 kb的坛紫菜hsp70基因,将其克隆至pMD18-T载体上测序.从测序正确的菌株中提取质粒,经限制性内切酶Sma Ⅰ和Not Ⅰ进行消化,将目的基因与真核表达质粒p181AINE连接,获得重组真核表达质粒p181-hsp70.通过菌落PCR、双酶切及测序等方法进行鉴定,结果表明重组真核表达质粒p181-hsp70构建成功.制备酵母感受态,将重组质粒电激转入酿酒酵母中,酵母菌落PCR结果显示电激转入成功.

Construction and Transformation of Eukaryotic Express Vector of hsp70 Gene from Porphyra haitanensis

Porphyra haitanensis,the seaweed with the great economic benefits,grows in the mutable intertidal environment,and is susceptible to the fierce changes of the temperature,osmotic pressure,irradiation and other stressors,so it often exposes itself to the adversity stresses.Heat Shock Protein 70(HSP70)as a highly conserved stress factor in the living organisms plays an important role in regulating the biological adaptation to the stress.It is helpful to learn the function of hsp70 in the stress resistance proc...