可诱导剔除选择标记基因的pBLCK载体构建

为了剔除冗余且有潜在安全问题的选择标记基因,本研究通过PCR扩增出ubiquitin启动子序列、lox P序列、Cre-GR融合基因和NPTⅡ抗性基因的编码区序列,采用酶切连接的方法将其引入载体p BRACT806,从而构建一个新的可诱导剔除选择标记基因及重组酶基因的表达载体p BLCK,测序结果显示其构建成功。该载体含有Gateway组件,可进行外源目标基因的高效重组整合;具有NPTⅡ基因,利于转基因植株的筛选;具有可诱导剔除外源基因的Cre-GR组件,诱导处于lox P位点之间的抗性标记基因NPTⅡ和Cre-GR融合基因的剔除,保留目标基因表达元件。此外,目标基因、Cre-GR融合基因和NPTⅡ基因都由ubiquitin强启动子控制,可在农作物中组成型超量表达外源基因。本研究为农作物转基因安全性研究提供了新的载体系统,具有一定的育种价值。

Construction of pBLCK Vector for the Inducible Deletion of Selectable Marker Genes

For excising redundant and potentially security concerned selectable marker genes in transgenic crops,the ubiquitin promoter,loxP sequence,coding regions of Cre-GR infusion gene and NPT Ⅱ selectable markder gene were amplified respectively.Afterwards,they were ligated into backbone vector pBRACT806 through restriction enzymes to construct a new vector for inducible deletion of selectable marker genes.The sequencing results showed that new plant expression vector pBLCK was constructed successfully.This vector includes Gateway cloning components for target gene recombination,NPTⅡ gene for screening positive transgenic plants,Cre-GR components for the inducible excisions of NPTⅡ and Cre-GR expression cassettes between two loxP sites,while keeping target gene in the host plant genome.Target gene with NPT Ⅱ and Cre-GR genes are under control of powerful ubiquitin booster,allowing the contitutive overexpression of these genes in crops.Hence,this study provide a new vector system to ensure the security of transgenic crops with the breeding value.