抗除草剂bar基因植物表达载体的构建与抗性功能的检测

Construction of Binary Plant Expression Vector Containing the Herbicide-resistant Bar Gene and Insect-resistant Bt Gene and Transformation of Cotton

根据GenBank上发表的抗除草剂bar基因序列设计引物,通过高保真DNA聚合酶进行PCR扩增,获得bar基因片段,连接在pMD18-T中间克隆载体上,用限制性内切酶XbaⅠ和BamHⅠ酶切后连接到pBI121上获得pBI121-bar。用HindⅢ内切酶切含Bt-4AB基因的质粒并与pBI121-bar重组,重组质粒命名为pBI121-bar/Bt。重组质粒导入农杆菌LBA4404工程菌中,转化新海24号棉花胚性愈伤组织。PPT抗性实验证明,构建的pBI121-bar/Bt载体获得抗除草剂抗性,并筛选出PPT临界筛选浓度为3.0mg/L。

According to designed primers of herbicide resistance bar gene sequence published on Gen- Bank,PCR amplified by high-fidelity DNA, the bar gene fragment were obtained, which were connected with the pMD18-T intermediate cloning on vector.The pBI121-bar was obtained when restrictive enzymes Xba Ⅰ and BamH Ⅰ enzymatic digestion were connected with pBI121.Hind Ⅲ endonuclease were cutted with Bt-4AB gene plasmid and were combined with pBI121-bar, the recombinant plasmid was named pBI121-bar/Bt.The recombinant plasmid were introduced into Agrobacterium LBA4404 engineering bacte- ria and were transformed to Xh24 cotton enbryogenic callus.The PPT resistance experiment showed that the built pBI121-bar/Bt vector had anti-herbicide resistance and screened PPT critical the screening concen- tration by 3.0 mg/L.