酸性蛋白酶pepB基因植物表达载体的构建及在玉米中的转化

以黑曲霉基因组DNA为模板, 利用聚合酶链式反应(PCR)技术扩增了酸性蛋白酶pepB基因序列，并将其克隆到pMD18-T Vector上，对重组子进行了PCR检测和限制性内切酶分析并测序。DNA序列分析表明：克隆片段长为1 340 bp，与已发表的pepB基因序列同源性达99.85%。将pepB基因片段插入到带有耐盐基因的表达载体pCAMBIA1301-BADH上，构建了无抗生素选择标记的重组质粒pB-pepB-35S，从而得到了植物表达载体，该载体利用耐盐基因BADH替代抗生素基因作为筛选标记。通过花粉管通道法将其导入玉米自交系，获得了转基因植株，并得到了转化植株的种子。

Construction of Expression Vector for pepB Gene of Acidic Protease and the Genetic Transformation in Maize

Genetic DNA of Aspergillus niger was used as total template to amplify the sequence of pepB gene of acidic protease by PCR technique, then PCR products were cloned into pMD18-T vector. The recombinant clone was detected by PCR technique and analyzed by restriction enzyme. Sequence analysis show that cloned fragment consisted of 1 340 bp and share 99.85% identity with the reported pepB. The cloned gene was inserted to the binary vector pCAMBIA1301-BADH with salt-tolerance BADH gene, which formed the recombinant plasmid pB-pepB-35S with antibiotic free selective marker, and a plant expression vector was thereby obtained. This expression vector used salt-tolerance BADH gene instead of antibiotic as a selective marker. Then we transformed them into different corn inbred lines by using pollen-tube pathway. Finally we got transgenic plants and we got transformed seeds.