以木糖异构酶基因xylA为选择标记的Gateway系统植物表达载体的构建

目的:构建以木糖异构酶基因xylA为筛选标记的无抗生素标记Gateway系统植物表达载体。方法:克隆大肠杆菌木糖异构酶基因xylA并用其替换植物表达载体pCAMBIA1301中的hpt基因,利用载体中的多克隆位点将Gateway Binary Vector(pH7WG2D)中酶切位点XbaⅠ和HindⅢ之间包括P35S、T35S、attR1、attR2和CmR-ccdB的片段重组入表达载体pCAMBIA1301中,构建表达载体pCAMBIA1301-xylA-GW,利用含有津田芜菁HY5基因片段的BP反应产物与载体进行LR反应,获得含有目的基因的植物表达载体pCAMBIA1301-xylA-HY5,并导入根癌农杆菌LBA4404中。结果:抗生素筛选及酶切和PCR鉴定表明成功构建了以xylA为筛选标记的无抗生素标记植物表达载体pCAMBIA1301-xylA-HY5。结论:利用木糖异构酶基因xylA结合Gateway克隆技术构建无抗生素标记植物表达载体,可简化、方便植物转基因表达载体构建。

Construction of Gateway Plant Expression Vectors with xylA Gene as Selection Marker

Objective：To construct plant expression vector with xylose isomerase gene xylA as the selection marker used in Gateway technology system.Methods：First,xylA gene was cloned from Escherichia coli,and was constructed into pCAMBIA1301 by substituting for hpt gene.Second,the fragment including P35S,T35S,attR1,at-tR2 and CmR-ccdB of the Gateway Binary Vector（pH7WG2D）was cut by XbaⅠand HindⅢand was recombinanted into the expression vector pCAMBIA1301-xylA.Third,the donor vector with HY5 genecloned from‘Tsuda’Turnip（Brassica campestris L.ssp.rapa）after BP reaction]was mixed with pCAMBIA1301-xylA-GW vector by LR reaction.Then,the recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 by electroporation.Results：The plant expression vector pCAMBIA1301-xylA-HY5 for Gateway technology system was successfully constructed by identification.Conclusion：The results showed that it is convenient to construct non-antibiotic marker plant expression vector by using xylA as selective marker and Gateway cloning technology.