含甘露糖异构酶基因植物表达载体的构建

Construction of Plant Expression Vector with manA Gene

目的:构建了以manA基因为选择标记的植物表达载体。方法:从大肠杆菌DH5α中克隆出manA基因,连接到质粒pCAMBIA1301的XhoⅠ位点,替换hpt基因,通过酶切和PCR检测了插入片段的正确性,使用XbaⅠ和HindⅢ酶切Gateway载体(pGWCBF)获得含有P35S-T35S-attR1-attR2-CmR-ccdB的结构域,将其插入到表达载体pCAMBIA1301的相应位点中,获得中间表达载体pCAMBIA1301-manA-GW,使用Gateway载体的BP反应与LR反应,将转录因子CBF基因片段整合到载体中。结果:酶切结果表明以甘露糖异构酶基因(manA)为选择标记的植物表达载体pCAMBIA1301-manA-CBF已经构建完成。结论:将构建好的载体用液氮冻融法转化到农杆菌中,可以用于葡萄的遗传转化研究,为将来获得安全的转基因抗寒植株奠定基础。

Objective:To construct the plant expression vector using manA gene as the selective marker.Method:The manA gene was amplified from the genomic DNA of Escherichia coli 'DH5α' by using specific primers containing XhoⅠenzyme site,the manA gene fragment was inserted into the plasmid pCAMBIA1301 to substitute the hpt gene,it was identified by enzyme digestion and PCR.Then,the Gateway Binary Vector(pGWCBF) was cut by XbaⅠ and HindⅢ to obtain the fragment including P35S-T35S-attR1-attR2-CmR-ccdB and inserted into the expression vector pCAMBIA1301.The donor vector with CBF gene was mixed with pCAMBIA1301-manA-GW by BP and LR reaction.Result:The recombinant vector pCAMBIA1301-manA-CBF was successfully constructed and verified by enzyme digestion.Conclusion:The vector can be transformed into Agrobacterium tumefaciens 'GV3101' by freeze thaw method and used in the grape genetic transformation,the following research could be done to obtain the cold tolerance transgenic plants with safe marker gene.