黄连对羟基苯基丙酮酸双加氧酶基因的植物表达载体构建

Construction of Coptis japonica 4-hydroxyphenylpyruvate Dioxygenase Gene Monocotyledon Expression Vector

目的:构建除草剂抗性基因黄连对羟基苯基丙酮酸双加氧酶的植物表达载体。方法:通过PCR从重组质粒pGWB2/Cjhppd中扩增出大小为1 300bp的目的片段,亚克隆到pGEM-T easy上。BamHⅠ和SpeⅠ双酶切pGEM-T Easy/Cjhppd和质粒pCambia1 301,回收得到1 300bp的Cjhppd基因片段和开环质粒pCambia-1301-UbiN,用T4连接酶连接,得到重组质粒,利用三亲法将其导入农杆菌EHA105中。结果:成功得到农杆菌EHA105的阳性菌落,菌落PCR扩增得到和预期大小一致的1300bpDNA片段。结论:成功将具有除草剂抗性的Cjhppd构建到了含有玉米泛素启动子Ubi和选择性标记基因Hpt的植物表达载体pCambia-1301-UbiN/Cjhppd,并导入根癌农杆菌EHA105中,可以用于水稻等单子叶植物的遗传转化。

Objective：The plant expression vector of Coptis japonica 4-hydroxyphenylpyruvate dioxygenase gene was constructed.Method：The expression vector contained a maize Ubi promoter and a selectable Hpt gene.The target gene Cjhppd was amplified by PCR from pGWB2/Cjhppd,and then was subcloned into vector pGEM-T easy.The recombinated plasmid pGEM-T easy/Cjhppd and pCambia1301 were digested by BamHⅠ/SpeⅠ at the same time,the two fragements were ligated into a plasmid vector pCambia-1301-UbiN/Cjhppd.After indentified by PCR and digestion with BamHⅠ/SpeⅠ,the recombined plasmid was introduced into Agrobacterium tumefaciens EHA105 by triparental mating method.Result：The plant expression vector contained a maize Ubi Promoter and a selectable Hpt gene was constructed successfully.Conclusion：The plant expression vector pCambia-1301-UbiN/Cjhppd with the herbicide resistance and increased content of vitamin E was constructed and introduced into Agrobacterium tumefaciens EHA105,which could be used in genetic transformation of monocotyledons.