摘要
金属硫蛋白 (MT)有α、β两个结构域 ,其中α结构域优先结合Cd2 + 、Hg2 + ,以α结构域代替 β结构域而得到的αα突变体在大肠杆菌构建及表达后 ,体外实验证明其具有同天然MT相似的稳定性 ,但结合重金属的能力稍强于天然MT。将αα突变体转入植物表达载体 ,以CaMV 35S为启动子 ,利用根癌土壤杆菌 (Agrobacteriumtumefaciens(SmithetTownsend)Conn)介导的叶盘法转化烟草 (NicotianatabacumL .cv.NC89)。Southern、Western证明了外源基因在烟草内的整合与表达。重金属抗性结果证明 :转αα突变体烟草在含 30 0 μmol/LCdCl2 的培养基中可正常生长 ,转天然MT的烟草在含 2 0 0 μmol/LCdCl2 的培养基中生长 ,而野生型烟草只能在含 75 μmol/LCdCl2 的培养基中生长。Cd2 + 含量及分布实验证明 :转基因烟草同天然烟草相比 ,根部吸收了大部分的Cd2 + ,从而降低了叶部Cd2 + 含量。
Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd 2+ and Hg 2+ . Mouse metallothionein mutant αα has been constructed and expressed in E.coli , which has the same stability as the nature one but has stronger affinity to heavy metals. To testify the result in vivo , αα mutant gene was cloned into plant expression vector pE3 under the CaMV 35S promoter. A transgenic tobacco was obtained by using leaf discs of tobacco ( Nicotiana tabacum L. cv. NC89) to Agrobacterium _mediated αα gene transfer. Southern blotting analysis indicated that the αα mutant gene was indeed integrated into the tobacco genome; Western blot indicated that the αα mutant gene was expressed in transgenic tobacco. It was also demonstrated that the transgenic tobacco with αα mutant gene have a little higher tolerance to heavy metals than that with natural MT gene. Moreover, the transgenic tobacco can accumulate more Cd 2+ in its roots than natural, so that, it can decrease the concentration of Cd 2+ in its leaves.
关键词
抗性
转基因
烟草
重金属
MT
土壤污染
metallothionein
αα mutant
plant expression vector
tolerance
