Gao Shang, Su Lei, Jia Hong-Ge, Guo Hong-Nian, Tian Ying-Chuan, Fang Rong-Xiang, Chen Xiao-Ying
State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Jan;23(1):157-60.
The commonly used plant constitutive expression vector pBI121 was modified by insertion of two directly orientated lox sites each at one end of the selectable marker gene NPTII and by replacing the GUS gene with a sequence composed of multiple cloning sites (MCS). The resulting plant expression vector pBI121-lox-MCS is widely usable to accommodate various target genes through the MCS, and more importantly to allow the NPTII gene removed from transformed plants upon the action of the Cre recombinase. In addition, the CaMV 35S promoter located upstream of the MCS can be substituted with any other promoters to form plant vectors with expression features specified by the introduced promoters. Provided in this paper is an example that an enhanced phloem-specific promoter of the pumpkin PP2 gene (named dENP) was used to construct an NPTII-removable phloem-specific expression vector pBdENP-lox-MCS. Moreover, to facilitate screening of selectable marker-removed gene and the composite sequence is flanked by lox sites. Thus the selectable marker-free plants can be visually identified by loss of GFP fluorescence. The above newly created plant expression vectors can be used to develop selectable marker-removable transgenic plants for a variety of purposes.
常用的植物组成型表达载体pBI121通过在选择标记基因NPTII的一端各插入两个同向lox位点，并将GUS基因替换为由多克隆位点（MCS）组成的序列进行了改造。所得的植物表达载体pBI121-lox-MCS可广泛用于通过MCS容纳各种靶基因，更重要的是，能使NPTII基因在Cre重组酶的作用下从转化植物中去除。此外，位于MCS上游的CaMV 35S启动子可被任何其他启动子取代，以形成具有所引入启动子指定表达特征的植物载体。本文提供了一个例子，即南瓜PP2基因的增强韧皮部特异性启动子（命名为dENP）用于构建一个可去除NPTII的韧皮部特异性表达载体pBdENP-lox-MCS。此外，为便于筛选去除选择标记的基因，复合序列两侧为lox位点。因此，无选择标记的植物可通过GFP荧光的丧失进行视觉鉴定。上述新创建的植物表达载体可用于开发多种用途的可去除选择标记的转基因植物。
