Abstract:

Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd2+and Hg2+. 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 ααgene 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 Cd2+ in its roots than natural, so that, it can decrease the concentration of Cd2+ in its leaves.

转金属硫蛋白突变体aa的烟草具有较高的重金属抗性
张晓钰 周慰 茹炳根*

（北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室，北京100871）

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Ｃｄ+２＋、Ｈｇ+２＋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ＣａＭＶ３５Ｓ为启动子，利用根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶盘法转化烟草（Nicotiana tabacum L.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300umol/L CdCl2的培养基中可正常生长，转天然MT的烟草在含200umoL/L)CdCl2的培养基中生长，而野生型烟草只能在含75umol/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Cd2+ ，从而降低了叶部Cd2+含量。
关键词： 金属硫蛋白；αα突变体；植物表达载体；抗性

Key words: metallothionein, αα mutant, plant expression vector, tolerance