在Genebank中找到花粉致死基因ZmAA1、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、终止子序列,并在目的片段的上下游设计増加同尾酶SpeI(NheI、XbaI)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-ZmAA1、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pCAMBIA3300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。
英文摘要:
Founding ZmAA1, Ms45, Mucronate(Mc) target gene and its Specific promoter、terminator sequence according to Genbank, And design to plus Restriction Endonuclease on the downstream of fragment of isocaudomers SpeI(NheI、XbaI), synthetic gene inserted into an cloning vector puc57,named puc-ZmAA1、puc-Ms45、puc-Mc. Target gene was inserted into an expression vector pCAMBIA3300 used traditional construction methods digested connection, and induced into Agrobacterium line LBA4404 and EHA105 by freeze-thaw method.Confirm plant expression vector was constructed successfully through digestion、PCR detection and Homologous analysis.
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