Arabidopsis Protoplast Isolation And Transient Expression Analysis Of Grape ERFs Regulating The GCC-box

The ethylene response factors (Ethylene Response Factor, ERFs) as a transcription factor is involved in the plant responses to the biotic and abiotic stresses. The expression of ERF could be induced by ethylene and plant diseases and then the ERF would recognize and bind to a cis-acting element GCC-box in the promoter of the downstream gene to regulate its expression. This will increase the capacity of the plant to defense the stresses. Therefore the study of the interactions between transcription factor ERFs and GCC-box will be very important.We have already cloned the open reading frames of 2 ERF genes from the cultivated grape Vitis vinifera, named VvERF2-1 and VvERF4-2 and 1 ERF gene from a wild grape V. aestivalis, named VaERF5. In this theses, we studied the regulation of ERFs to the GCC-box in the promoter region of a recombined expression vector which containing a firefly luciferase reporter plasmid pGCC by transient expression system of PEG-Ca co-transforming Arabidopsis chloroplast. The fluorescence was measured in the transient expression system to investigate the reactions between the ERFs and GCC-box in the promoters respectively. The results showed that:1) The best yield and quality of the isolated protoplasts could be obtained from the three to four weeks old and middle part of the leaf blade of Arabidopsis plants cultivated in artificial soil and the optimum centrifugal force should be at 40-60 g and with one or two brake value.2) The optimum concentration of PEG transformation buffer is 40%. In this concentration, the protoplast transformed kept round and fewer broken.3) WI protoplast culture medium is much better than W5 for culturing the transformed chloroplasts4) No fluorescence was detected in the transient expression system of VvERF2-1 plus the GCC-box co-transformation.5) A significant enhancement of fluorescence was detected in the transient expression system of VvERF4-2 plus the GCC-box co-transformation.6) No fluorescence was detected in the transient expression system of VvERF5 plus the GCC-box co-transformation.