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[目的]构建以甘露糖为筛选剂的抗旱、抗盐碱植物表达载体,进一步选育无标记的抗逆作物品种。[方法]利用腊梅花水通道蛋白CpTIPcDNA和大肠杆菌pmi基因构建植物表达载体,将抗逆基因与甘露糖正向选择系统结合。pmi基因植物表达载体(pPMI)的构建:用XhoⅠ酶切植物表达载体pCAMBIA2301,切除Kan基因,并用CIP酶进行去磷酸化,然后与XhoⅠ酶切后PCR产物连接转化。腊梅花水通道蛋白CpTIP基因甘露糖筛选体系植物表达载体(pPMI::CpTIP)的构建:用KpnⅠ和XbaⅠ酶切pPMI植物表达载体和克隆载体,并将酶切后pmi植物表达载体与腊梅花水通道蛋白CpTIP基因连接转化。转化鉴定均按Sambrook等的方法进行。[结果]成功构建了腊梅花水通道蛋白CpTIP基因甘露糖筛选体系植物表达载体pPMI::CpTIP。[结论]所构建的载体结合了抗逆基因和甘露糖正向选择系统的优点,将抗逆基因与环境友好型筛选体系很好的结合起来。
[Objective] The research aimed to construct a drought-resistant and salt-resistant plant expression vector with mannose as the screening agent and further to breed unmarked anti-stress crop varieties. [Method] The plant expression vector was constructed by using CPTIP cDNA of Plum blossom and pmi gene of Escherichia coli to combine the resistance gene with mannose positive selection system. Construction of pmi gene plant expression vector (pPMI): The plant expression vector pCAMBIA2301 was digested with XhoI, the Kan gene was excised and dephosphorylated with CIPase, and then ligated with the PCR product after XhoI digestion. The construction of the plant expression vector (pPMI :: CpTIP) of the plum blossom aquaporin CpTIP gene mannose screening system: The pPMI plant expression vector and the cloning vector were digested with Kpn I and Xba I, and the pmi plant expression vector was digested with the bloom flower water Channel CpTIP Gene Conjugation and Transformation. Transformation identification were performed according to Sambrook et al. [Result] The plant expression vector pPMI :: CpTIP of mandelose screening system of CpTIP gene was successfully constructed. [Conclusion] The constructed vector combined with the advantages of the anti-retrogradation gene and the mannose forward selection system, and well combined the anti-retrogradation gene and the environment-friendly screening system.