玉米隐性核不育保持系表达载体构建

Expression Vector Construction of Recessive Genic Male Sterility Maintainer Line of Maize

在Genebank中搜索花粉致死基因Zm AA1、花粉育性恢复基因Ms45、粉质胚乳突变基因Mucronate及各基因特异调控因子和终止子序列,并在目的片段的上下游设计增加同尾酶Spe I(Nhe I、Xba I)和酶切后具有各种类型的DNA片段的Esp3I酶切位点,基因合成构建到克隆载体puc57上,命名为puc-Zm AA1、puc-Ms45、puc-Mc。采用传统酶切连接的方法将目的片段构建到植物表达载体p CAMBIA3300上,并用热击法将重组质粒导入农杆菌LBA4404及EHA105中,酶切、PCR检测及核苷酸序列测定证明,植物表达载体构建成功。

According to Genbank, Zm AA1, Ms45, Mucronate（Mc） target gene and its specific promoter and ter-minator sequence were found, and design to plus restriction endonuclease on the downstream of fragment of isocau-domers Spe I（Nhe I, Xba I）, synthetic gene inserted into a cloning vector puc57, named puc-Zm AA1, puc-Ms45 andpuc-Mc. Target gene was inserted into an expression vector p CAMBIA3300 used traditional construction methodsdigested connection, and induced into Agrobacterium line LBA4404 and EHA105 by freeze-thaw method. Confirmplant expression vector was constructed successfully through digestion, PCR detection and homologous analysis.