小鼠金属硫蛋白突变体αα-cDNA在烟草中的表达及转基因烟草对Cd^(2+)抗性的提高

Expression of the Mouse Metallothionein Mutant αα-cDNA Improving Cadmium Resistance in Transgenic Tobacco

金属硫蛋白是一类小分子量的金属结合蛋白 ,有 β,α两个结构域 .用另一个 α结构域取代金属硫蛋白的 β结构域 ,得到突变体 αα.通过 PCR法在 αα- c DNA翻译起始密码子 ATG前加入植物偏爱的碱基组合 AACA,构建 Ca MV双 35S启动子驱动的 αα- c DNA植物表达载体 p GPTVd35S-αα,此载体以抗除草剂基因 ( bar)作为选择标记物 .用根癌农杆菌介导的叶盘法对栽培种烟草 NC89进行转化 .Southern和 Western印迹分析表明突变体αα- c DNA已整合进入烟草基因组并且可以正常表达 .Northern印迹结果显示突变体αα- c DNA在烟草根部的表达强于叶部 .烟草根、茎、叶Cd2 +含量分析表明 ,转基因植物与对照相比 ,Cd2 +更多地集中在根部 ,在一定程度上降低了叶片中的 Cd2 + 含量 .Cd2 + 抗性实验进一步证明突变体αα- c DNA的表达提高了转基因烟草对 Cd2 + 的抗性 :转基因烟草在 2 0 0μmol/L Cd Cl2 条件下生长正常 ,在 40 0μmol/Cd Cl2 培养基内也可以存活 .而 1 0 0μmol/L Cd Cl2 即成为对照烟草的致死剂量 .

Metallothionein is a metal binding protein with small molecular weight,which has β and α two domains.Mouse Metallothionein mutant αα has been constructed by replacing the β domain of the metallothionein with the other α domain.Some nucleotides beside ATG were changed to AACAATG by PCR method,which were favored in plants.CaMV 35S 35S/αα cDNA expression vector pGPTVd35S αα was constructed which had herbicide(PPT) resistance gene bar as a selectable marker.The chimeric gene via Agrobacterium mediated transformation was introduced into the tobacco( Nicotiana tabacum L.c.v.NC89).According to Southern blot and Western blot analysis of some putative transformants,it was found that the introduced αα cDNA was integrated into tobacco genome and could be expressed.Northern blot analysis indicated that the transcripts of αα cDNA in roots were stronger than these in leaves.Determination of Cd 2+ concentration and its distribution inside the plants demonstrated that the transgenic tobacco could accumulate more Cd 2+ in its roots than the control,which could reduce the content of Cd 2+ in the leaves.The experiment of the resistance to Cd 2+ proved that the expression of αα cDNA in tobacco enhanced the plant tolerance towards Cd 2+ .Under 200 μmol/L CdCl 2,plants with normal growth were observed.Transgenic tobacco could even live in the medium containing 400 μmol/L CdCl 2,while 100 μmol/L CdCl 2 became toxic during 30 days growth period.