This method can help answer key questions in the plant molecular and cell biology field such as, how can we proteins in the cell. The main advantage of this technique is the high efficiency of co-expressing cellular fusion proteins in one expression vector. Demonstrating the procedure will be Guitao Zhong, a graduate student from our lab.
To begin, design the primers for molecular cloning of DNA fragments as described in the text protocol. Amplify DNA fragments that are necessary for the construction of the semi-independent protein expression cassettes by standard PCR reactions with their corresponding primers and high-fidelity polymerase. These include the promoter, fluorescent reporter, target gene, and terminator.
Examine the quality of the first round PCR products by looking for DNA degradation and contamination with DNA electrophoresis using a 1%agarose gel. Quantify the PCR products with a spectrophotometer. The ratio between the readings at 260 and 280 nanometers of the PCR products should be between 1.6 and 1.8.
Mix the DNA fragments designed for the same protein expression cassette together in one PCR tube to a final volume of five microliters. About mixing DNS from different expression cassette data. Since equal reduces the efficiency of DNA assembly, due to increasing numbers of DNA molecules that need to be linked.
Now, add 15 microliters of 2x master mixture to the 5 microliter DNA mixture and incubate at 50 degrees celsius for 60 minutes. Amplify the entire semi-independent protein expression cassette by a second round of PCR. Use 0.5 to one microliters of the product from the first round isothermal assembly reaction as the template in the outermost primers.
Use one unit of high-fidelity polymerase in a 50 microliter reaction volume for 30 cycles, followed by a final extension at 68 degrees celsius for five minutes. Next, linearize the final protein expression backbone vector POC 18, and vector CAMBIA 1300, by adding four units of small one into a final 10 microliter reaction volume. These vectors are designed for protein transient expression and genetic transformation.
Incubate the restriction digest for one to two hours at 25 degrees celsius. Following digestion, inactivate the restriction enzyme by incubating at 65 degrees celsius for 20 minutes. Next, mix equimolar DNA molecules of protein expression cassettes and linearized final vector into a final reaction volume of five microliters.
Finally, perform the second round of DNA recombination by mixing the reaction with 15 microliters of 2x master buffer, and incubating at 50 degrees celsius for 60 minutes. To prepare tobacco BY-2 suspension cells for bombardment, filter-collect 30 milliliters of 3-day cultured BY-2 cells onto a piece of 70 milliliter autoclaved filter paper via a vacuum pump, by setting the vacuum pressure to 40 millibar. Meanwhile, add several drops of BY-2 cell liquid cultural medium into a petri dish.
Transfer the filter paper with the BY-2 cells on it to the petri dish. To prepare arabidopsis juvenile plants for bombardment, prepare seven-day-old sample plants as detailed in the text protocol. Then, transfer them into a rectangle in the center of a new half-strength MS medium plate to increase the efficiency of bombardment.
Take care to avoid overlapping the plants when transferring and placing them onto the plate. Add several drops of half-strength MS liquid medium on the surface of the plants or tissues to preserve moisture and prevent drying the plants out during the remaining steps. To coat gold particles with plasmid DNA, first vortex the gold microcarrier solution thoroughly for three minutes.
Now, add 25 microliters of gold particles to a new 1.5 milliliter tube, and vortex for 10 seconds. Add 10 microliters of 25.46 milligrams per liter spermidine and vortex for 10 seconds. Next, add five microliters of one microgram per microliter plasmid DNA, and vortex for three minutes.
Finally, add 25 microliters of 277.5 milligrams per liter calcium chloride solution, and vortex for one minute. Spin down the gold microcarriers using a bench-top centrifuge at maximum speed for five seconds. Following centrifugation, carefully pipe it out the supernatent without disturbing the pellet.
Next, wash the pellet with 200 microliters of absolute ethanol, and re-suspend the pellet by vortexing for five to 10 seconds. Once gain, spin down the gold microcarriers at maximum speed for five seconds, and remove the ethanol. Re-suspend the gold particles in 18 microliters of absolute ethanol.
Then, aliquot six microliters of particle suspension onto the middle of three microcarriers and let them air dry. To transfer the DNA into the cells and plants via particle bombardment, first set the particle delivery system as detailed in the text protocol. Then, bombard the cells or plants on the agromedium plate for three times at three different positions.
Keep the bombarded cells and plants in the dark in the plant growth chamber for six to 72 hours prior to observation of fluorescent signals. Set the plant growth chamber to 24 hours dark and 22 degrees celsius. Transfer the juvenile plants or suspension cells onto a conventional glass slide and gently put a cover slide on the top for imaging by standard confocal laser scanning microscopy.
Using the settings listed in the text protocol, excite GFP tagged proteins at 485 nanometers, and detect fluorescence at 525 nanometers. For RFP tagged proteins, excite at 585 nanometers and detect at 608 nanometers. Finally, calculate the collocalization ratio of fluorescent signals as described in the text protocol.
Co-expression of arabidopsis VSR-2 and arabidopsis SCAMP4 in tobacco BY-2 cells was achieved via particle bombardment and show correct localizations. RFP arabidopsis VSR-2 displays a punctate pattern, which was distinct from the plasma membrane localization of arabidopsis SCAMP4-GFP, with some cytosolic punctate dots. Moreover, arabidopsis transgenic plants that co-express arabidopsis SCAMP4-GFP and RFP arabidopsis VSR-2 were generated via agrobacteria-mediated transformation.
The subcellular localizations of co-expressing the two chimeric proteins in root and root hair cells is shown. Consistent with pervious studies, treatment of transgenic arabidopsis caused RFP arabidopsis VSR-2 labeled prevacualar compartments, forming a small ring-like structure. Whereas treatment with prefoldin A induced arabidopsis SCAMP GFP labeled transgold G-network aggregation.
As a negative control, little autofluorescent signal is observed in tobacco BY-2 cells and arabidopsis root and root hair cells by applying the same settings of image collection. This method can provide insight into the subcellular localization of the proteins. It can also be applied to the study of protein in direction.
After its development, this technique paved the way for researchers in the field of plant cell biology. To conveniently export the subcellular localizations and spacial interaction of proteins in the living plant cell. Following this procedure, other methods like PET mediated transformation and genetic crossing can be performed in order to answer additional questions like, how to co-express several fusion proteins in plants.
Don't forget that working with bombardment can be extremely hazardous and precautions such as eye protectors should always be taken while performing this procedure.
