植物の細胞や細胞小器官の壁を越え遺伝子やタンパク質を輸送するために、ペプチド由来方法

The overall goal of this peptide-derived method is to transport genes and proteins across cellular and organellar barriers in plants. This method can help answer key questions through the modification of plants via rationally designed peptide conjugates which deliver various genes and proteins to mediate the expression of specific genes. The immediate advantage of this technique is that it employs a simple syringe-assisted transfection procedure of plant leaves that is based on peptides which can transcend cellular and organellar boundaries.

We developed this simple, effective, and novel plant transformation method because conventional methods are costly, not applicable to some plant types, and unable to target intracellular organellars. Jo-Ann Church will be demonstrating the procedure with Yoko Horii, a technician from my laboratory. Prepare the peptide plasma DNA formulation as described in the text protocol.

When the peptide is added to the plasma DNA, a cloudy solution will form. The mixture becomes clear upon dilution with ultrapure water to the final volume specified in the text protocol. Transfer 800 microliters of the diluted solution into a cuvette for dynamic light scattering analysis.

Determine the hyrodynamic diameter and polydispersity index of the formed complexes with a zetananosizer. Using a 633 nanometer helium neon laser at 25 degrees celsius with a backscatter detection angle of 173 degrees. Following size measurements, transfer the solution into a folded capillary cell for zeta potential measurements at the default parameters of the dielectric constant, refractive index, and viscosity of water at 25 degrees celsius.

To observe the complex solution by atomic force microscopy, deposit 10 microliters onto the freshly exposed cleaved surface of a mica sheet and leave the mica to air dry overnight in a covered plastic Petri dish. Acquire an image of the complexes in air at 25 degrees celsius using a silicon cantilever with a spring constant of 1.3 newtons per meter in tapping mode. Use three week old Arabidopsis thaliana grown under a cycle of 16 hours light, 8 hours dark, at 22 degrees celsius.

Choose at least three leaves for transpection to serve as a triplicate for the quantification of gene expression. Load a one-millileter needleless plastic syringe with 100 microliters of complex solution for the transfection of one leaf. Then, position the tip of the syringe on the abaxial surface of the leaf.

Press the syringe tip against the leaf slightly, and depress the syringe plunger slowly while exerting a counterpressure with the index finger of a latex gloved hand from the opposite side. Successful infiltration can be observed as the spreading of a water-soaked area in the leaf. Label the infiltrated leaves for ease of identification.

Incubate the peptide plasma DNA-transfected plant for 12 hours in a growth chamber. To evaluate the transfection efficiency, first excise the whole transfected leaf. For transfection experiments using the Renilla Luciferase reporter vector, determine the transfection efficiency quantitatively using a Renilla Luciferase assay kit.

First, place each excised leaf in a 1.5 millileter microcentrifuge tube. Add 100 microliters of 1X Lysis Buffer per tube. In the same manner, prepare lysates of non-transfected control leaves in triplicate.

Grind the leaf using a homogenization pestle and incubate the resultant lysate at 25 degrees celsius for six to 10 hours. Following incubation, centrifuge the lysate at 12, 470 times g in a microcentrifuge for one minute. Transfer 20 microliters of the cleared lysate to a well in a 96-well microplate.

Use the remaining volume for quantification of total protein concentration using a BCA protein assay kit according to the manufacturer's protocol. Next, add 100 microliters of one times Renilla Luciferase assay substrate into the well and mix by slow pipetting. Place the microplate in a multi-mode microplate reader and initiate measurement.

For transfection experiments using a green fluorescent protein reporter vector, observe the fluorescence using a confocal laser scanning microscope. First, cut the edges of a whole leaf to aid the removal of air spaces. Then, remove the plunger from a 10 milileter syringe and place each excised leaf or leaf section in the syringe.

Replace the plunger and push it gently to the bottom of the syringe without crushing the leaf. Draw water into the syringe until it is approximately half-filled. Point the syringe upwards and push on the plunger to remove air from the syringe through the tip.

Then, cover the tip of the syringe and pull the plunger down slowly to expel air from the leaf. Repeat this process several times until the leaf appears translucent. Next, cover the surface of a microscope slide with adhesive tape.

Cut a square area on the tape large enough to accommodate the leaf sample using a blade, and peel the square piece of tape off with forceps to create a specimen chamber. The tape will serve as a spacer between the slide and cover slip. Place the leaf in the chamber with the abaxial surface facing upward and fill the remaining chamber area with water.

Seal the leaf within the chamber with a glass cover slip and secure the edges of the cover slip with adhesive tape. Examine the leaf sample using a confocal laser scanning microscope under a 40x objective or a 63x water immersion objective. GFP fluorescence can be visualized at an excitation wavelength of 488 nanometers.

An optimal preparation of the peptide plasma DNA formulation at a peptide-to-DNA ratio of 0.5 should have a mean diameter of approximately 290 nanometers and a low polydispersity index of approximately 0.3 which indicates a uniform size distribution. Additionally, the complexes should exhibit a zeta potential of approximately 30 millivolts. A representative atomic force microscope image of the peptide plasma DNA complexes is shown here.

Homogenous globular complexes were observed. Using the peptide plasma DNA formulation, nuclear-targeted delivery and expression of plasma DNA can be achieved in Arabidopsis thaliana with an estimated Renilla Luciferase per miligram value of approximately 100, 000. Microscopic observation was performed on leaves treated with complexes prepared using plasma DNA, encoding the GFP reporter to assess gene expression.

The images revealed that in cells transfected with non-targeted peptide plasma DNA complexes, diffuse green fluorescence corresponding with GFP expression was clearly observed and found to localize to the cytosol. After its development, this technique paved the way for researchers in the field of plant biotechnology to deliver various protein and nucleic acid-based cargoes into living plants. After watching this video, and reading the text protocol, you should have a good understanding of how to prepare, characterize, and apply various optimized fomulations for each type of cargo, which include the plasma DNA, double-strand DNA or RNA, and protein.