The overall goal of this procedure is to use a Gus reporter system to analyze the potential death properties of specific genes. Genes of interest are cloned into a Gus cassette and grown in the appropriate agrobacterium vectors under a 35 s promoter. These vectors are then transformed into Agrobacterium strain, LBA 44 0 4, and are mixed with a culture containing a Gus expression cassette to infiltrate Nick Oceana Hamana leaves.
Gus activity can be assessed visually by histochemical staining, or quantitatively by Fluor metric mug assay. The main advantage of this technique over existing methods like DNA laddering or tunnel assay is commonly used in mammalian systems, but difficult to reproduce in plants, is that this is sensitive, rapid plant-based transient assay that takes advantage of the simple fact that live cells are required for gas expression to occur. Demonstrating the procedure today will be Hua RAOs.
A post-doc research associate in our lab Infiltration requires fully expanded healthy leaves of three to six week old nicotiana hamana plants grown at 25 degrees Celsius. Plan to carry out this protocol over eight days. On day one streak grates with glycerol stocks of agrobacterium tumor fassan containing the vectors for the cell death assay and the vector with the Gus expression cassette also always include an empty vector as a negative control or stuffer when multiple culture combinations are being tested.
Incubate the plates at 28 degrees Celsius for two days. On the third day, inoculate two milliliters of selective LB with a single colony from each plate. Incubate each culture by shaking at 28 degrees Celsius for 24 hours at 200 RPM until maximum growth density is reached.
After 24 hours of incubation, completely resuspend the culture by pipetting if there is clumping, and increase the total volume for each culture to 10 milliliters with fresh selective LB to induce agrobacterium virulence genes. Add acetone to 25 micromolar and incubate each culture with shaking at 28 degrees Celsius for another 16 hours. The next step is to centrifuge each culture at 4, 000 Gs for 10 minutes at room temperature and Resus, suspend the pellet in 10 milliliters of infiltration medium.
Do not add acetone, centrifuge the cultures again, but now Resus. Suspend each pellet in five milliliters of infiltration medium containing 100 micromolar acetone. Now measure the OD at 600 nanometers.
Concentrations as low as 0.1 are acceptable, but if greater than 0.9, then dilute to 0.9 with infiltration, medium and acetone prior to infiltration. Incubate the cultures once more at 28 degrees Celsius with shaking for three hours after the incubation. Mix the experimental cultures and control cultures in a one-to-one ratio with the culture containing the Gus cassette.
For each culture, use a one milliliter needleless syringe to infiltrate the ABA seal or underside of newly emerging leaves. The infiltration volume is not important. When a water soaking area forms enough has been added, infiltrate both the negative control and the gene to be assay on opposite leaves on the same plant, then infiltrate two more plants in the same way.
So there are triplicate plants for each culture. Three days after infiltration, it is time to collect the leaves and assay for gust protein expression. Begin the histochemical analysis by vacuum infiltrating the entire leaf with xlu substrate until it is completely soaked.
Now, incubate the leaves in darkness at room temperature overnight or until distinct blue Staining appears when blue staining is evident. Rinse the leaves in distilled water and incubate them in 70%ethanol overnight until their chlorophyll is removed. The next day, rinse the leaves off and distilled water and assess the GUS expression levels.
To quantify Gus expression, a substrate mug is converted into a fluorescent product mu, which can be measured begin by grinding infiltrated leaf discs in a mortar using liquid nitrogen. Then add 100 microliters of GUS extraction buffer while keeping the sample on ice. Now centrifuge the suspension at 14, 000 GS per five minutes at four degrees Celsius, and collect the supernatant containing the total soluble proteins, which includes Gus.
Measure the total soluble protein concentration using an NanoDrop and adjust the concentration to 100 micrograms of total protein for each sample in Gus extraction buffer. Now, add mug substrate in Gus extraction buffer to each sample to a final concentration of two millimolar mug. A total volume of 100 microliters per sample should be sufficient mixed by pipetting and incubate the reaction for an hour at 37 degrees Celsius.
After an hour, stop the reaction by adding 800 microliters of gusts stop buffer. To quantify the mu 100 microliters of each sample is loaded onto a microtiter plate, which is analyzed in a plate reader fluorescence at 365 nanometers excitation 455 nanometers emission wavelengths is measured and compared to a standard curve, Guss activity can then be reported as M of mu per microgram of total soluble protein per minute reaction. Various gust levels are detected depending on the construct used when the GUS expression cassette is co infiltrated with a negative regulator of cell death or an anti-death gene.
High gust levels are observed by comparison. Gus levels in the empty vector control are much lower. Conversely, when a positive regulator of cell death or a pro-death gene is used, a market reduction in Gus expression is observed compared to the empty vector control.
Once mastered, this technique can be done in approximately one week. While attempting this procedure, it is important to remember that this technique is used as an initial step to determine the potential of specific genes. Additional work needs to be done to confirm these initial results and to determine the type of cell that involved.
