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采用Trizol剂试提取玉米胚乳总RNA,设计相关引物,采用反转录PCR方法克隆腺苷二磷酸葡萄糖焦磷酸化酶编码基因bt2和sh2,并克隆玉米胚乳特异表达的isa基因启动子,利用酶切连接方法,构建bt2和sh2基因的单子叶植物胚乳特异表达载体。结果表明,bt2和sh2基因正向插入胚乳特异的ISA启动子和T-nos终止子之间,成功构建p3301-ISA-bt2-T35S和p3301-ISA-sh2-T35S植物胚乳特异表达载体。
Total RNA was extracted from corn endosperm by Trizol reagent. The primers were designed. The adenosine diphosphate glucose pyrophosphorylase coding genes bt2 and sh2 were cloned by reverse transcription PCR. The isa gene promoter specifically expressed in endosperm of corn was cloned. Cut connection method to construct bt2 and sh2 gene monocotyledonous endosperm specific expression vector. The results showed that the bt2 and sh2 genes were inserted into the endosperm-specific ISA promoter and T-nos terminator in the forward direction, and the endosperm-specific expression vectors p3301-ISA-bt2-T35S and p3301-ISA-sh2-T35S were successfully constructed.