ABSTRACT
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.
Subject(s)
Attachment Sites, Microbiological , Genetics , Binding Sites , Genetics , Cloning, Molecular , Gene Knockout Techniques , Methods , Genes, Plant , Genetics , Genetic Markers , Genetics , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Metabolism , Integrases , Genetics , Metabolism , Plants , Genetics , Plants, Genetically Modified , Genetics , Recombination, GeneticABSTRACT
Using total DNA isolated from Amaranthus caudatus as the template, a DNA fragment of about 700bp upstream of the coding sequence of Amaranthus caudatus agglutinin (ACA) gene was amplified by TAIL-PCR and cloned. To examine the regulatory function of this DNA fragment, it was inserted into a plant expression vector containing GUS gene to substitute the CaMV 35S promoter and the resulted recombinant plasmid was designated as pBpAG. The expression vector pBpAG was transferred to different tissues of plants, via Agrobacterium-mediated transformation in vacuum condition. Transient expression of GUS in the transformed tissues was detected by histochemical GUS staining and the results showed that the GUS activity was expressed specifically in seeds. These preliminary results indicate that this DNA fragment upstream of the ACA coding sequence could very possibly be a promoter with seed specificity. Some putative cis-elements within the promoter were discussed.