金属硫蛋白突变体的植物高效表达载体的构建及其在烟草中的表达

金属硫蛋白有α、β两个结构域（ｄｏｍ ａｉｎ），其中α结构域优先结合Ｃｄ２＋ 和Ｈｇ２＋ ．小鼠αα突变体在大肠杆菌中已经构建并得到表达，其转基因植株已得到，可在Ｃｄ３００（３００ μｍ ｏｌ／Ｌ）中生长．为了进一步提高外源基因在烟草中的表达量，首先用ＰＣＲ 的方法设计引物，在基因翻译起始密码子ＡＴＧ 附近加入植物偏爱的碱基组合ＡＡＣＡＡＴＧ．另外，将该突变体基因插入具有双３５ Ｓ（ＣａＭＶ３５Ｓ）强启动子的植物双元表达载体ｐＧＰＴＶｄ３５Ｓ－ＢＡＲ中，获得了带有αα突变体的植物双元表达载体．通过农杆菌介导的叶盘转化法转化烟草ＮＣ８９，获得了抗除草剂的转基因植株．经ＰＣＲ－Ｓｏｕｔｈｅｒｎ 和蛋白Ｄｏｔ－ｂｌｏｔｔｉｎｇ 检测，证明了αα突变体在烟草中的嵌合与表达．抗重金属实验证明转基因烟草可以在Ｃｄ４００（４００ μｍ ｏｌ／Ｌ）中生长．

Construction of High Efficient Expression Vector of Metallothionein Mutant in Plant and its Expression in Tobacco

Metallothionein has two domains,α domain and β domain.α domain preferred to bind Cd 2+ and Hg 2+ .Mouse metallothionein mutant αα had been constructed and expressed in E.Coli ,and its transgenic tobacco had been obtained.To improve the expression level of foreign gene two approaches were applied:first,designing primers and changing some nucleotides besides ATG to AACAATG which were favored by plant in the assay of site directed mutation;second,inserting this mutant gene into plant expression vector pGPTVd35S BAR which had strong promoter with double 35S of CaMV.Now this vector with metallothionein mutant had been obtained.Leaf discs of tobacco NC89 were infected by Agrobacterium which contained plant expression vector and bar resistant plants from individual transformation event were obtained.Tested by PCR,PCR Southern and protein Dot blotting,some of them were positive.Resistance to heavy metal proved that these transgenic tobacco could even live in the medium containing 400 μmol/L Cd 2+ .