Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases.
The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.
Jia
Fan
F
Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, USA. fan.jia@ttu.edu
Gampala
Srinivas S L
SS
Mittal
Amandeep
A
Luo
Qingjun
Q
Rock
Christopher D
CD
D000040:Abscisic Acid / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics*; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D030821:Plants, Genetically Modified; D000071636:Protein Phosphatase 2C; D011523:Protoplasts / Q000378:metabolism; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D000040:Abscisic Acid; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C