The overall goal of this procedure is to demonstrate a method for growing an inducible flax variety under conditions that result inheritable genomic variation. Furthermore, genomic changes are monitored and the growth of the induced plants in subsequent generations is observed under the various environments. To accomplish this, flax is grown under a variety of inducing conditions.
Leaf and Maris stem tissue is then collected at various times during the growth of the plants under the various environments. Next, DNA and RNA are isolated from the tissue samples. The final step of the procedure is to perform PCR using the DNA samples and Q-R-T-P-C-R using the isolated RNA to identify genomic changes and gene expression changes resulting from growth under different environmental conditions.
Ultimately, results can be obtained to show the genomic response to growth conditions with the appearance of the LIS one insertion element at a specific point in the genome and expression differences associated with the presence of that element as visualized by PCR and R-T-P-C-R performed with nucleic acid isolated from the tissues of treated plants. Hi, I am Chris Cull from the Department of Biology at Case Western Reserve University. I'm Cory Johnson from the Cull Lab.
And I'm Tiffany Moss, also from the cull lab. And I'm Marshall Lucas. An undergraduate in the summer program for undergraduate research at Cull lab.
We use this procedure in our laboratory to study changes in the genome and gene expression resulting from variations in the growth environment. So let's get started. To grow flax under inducing conditions begin by filling five inch pots with soil and firming.
Next plant, three flax seeds per pot, one quarter inch deep. Then from the soil over the seeds for the non inducing control conditions. Water the plants with 100 milliliters of one 10th strength miracle grow solution.
Treat the plants under high nutrient conditions with 100 milliliters of full strength. Miracle grow. Finally apply.
Only tap water to the low nutrient plants. Grow the plants under natural light during the summer as the plants grow. Water all the plants by an automatic spray system twice a day for five minutes.
Apply the growth conditions to the plants once a week. Collect the leaves into mari stems at weekly intervals. Six leaves are collected for DNA extraction, and three me stems are needed for RNA extraction following collection, place the plant material in a cryo vial, quickly freeze it in liquid nitrogen and store the samples at minus 80 degrees Celsius until extracted.
The three genotypes grow at different relative rates depending on the conditions. Under control conditions, the inbred flax variety storm or the PL line grows best. Several lines were derived from this parent with a large or L variety being more vigorous than the small or S variety.
However, under low nutrients S grows better than either L or pl. And under high nutrient conditions, L grows better than pl, which only grows slightly better than S.A comparison between each of the lines grown under the three differing treatments reveals that PL grows best under control conditions. L grows best under high nutrient conditions, and S grows similarly under both high nutrient and control conditions.
These data show that the three lines can be differentiated based on their growth under the three environments. While PL and L grow better under specific environments, S grows about the same in all three conditions. S is not able to take advantage of improved nutrient conditions, but is better able to grow under very low nutrient conditions.
For DNA extraction add buffer, AP one from the Qiagen, DNEZ plant, DNA prep kit to six leaves with sterile sand in a micro fuge tube, grind the tissue with a micro pestle until no clumps are visible. Continue the extraction by following the kit instructions as directed to begin RNA extraction transfer, three maris stems plus some surrounding leaves from a storage cryo vial to a micro fuge tube. Add sterile sand to the tube and grind the tissue using a micro pestle until finely ground.
Vortex the sample for 15 seconds to aid lysis, then spin for one minute at 13, 000 RPM to collect sand and any debris. Add the SNAT to the spin column and continue with RNA preparation per manufacturer's instructions. Once the RNA is extracted from the tissue transfer 10 XDNA buffer at one 10th of the RNA sample volume to the reaction.
And at 30 units of D Ns, incubate the reaction at 37 degrees Celsius for 30 minutes, followed by 70 degrees Celsius for five minutes. Perform reverse transcription using the applied biosystems RNA to CD NA kit. As per manufacturers and S instructions, incubate the reaction at 37 degrees Celsius for one hour, and then at 95 degrees Celsius for five minutes.
Finally, run five microliters of the CDNA on a 1.5%AROS gel to determine yield. Once the flax CD NA has been prepared, it is used for QPCR. Carry out QPCR using an A BI Step one system.
Perform all assays in triplicate for each run, and use the housekeeping Gene Acton as a copy number control to prepare the reactions for QPCR. Add one microliter of the appropriate primer pair to each tube. Dilute the CD NA to the appropriate concentration and add nine microliters to each tube.
Finally, add 10 microliters of the two x cyberg green PCR master mix to each tube. Next, set up the amplification program following amplification. Calculate the relative expression levels from the cycle number for threshold, which is determined by the default parameters in the step one software and confirmed by inspection of the amplification curves.
PCR amplification from the DNA, isolated from leaves at various positions along the stem demonstrates the appearance of a single copy 5.7 kilobase, DNA fragment termed line insertion sequence, or LIS one when the plants are grown under inducing conditions. LIS one appears when PL is grown under low nutrient inducing conditions and eventually becomes homozygous.Here. Leaf samples one through six were isolated at weekly intervals with sample one being the earliest after sewing DNA was then extracted from the samples.
PCR amplification reveals the presence of both termini of LAS one in all the samples except the first. This corresponds with the disappearance of the unins inserted site and no evidence of it by the fifth leaf QPCR. Results show that the presence of LAS one or other genomic changes associated with the induction affects the expression of genes in the immediate vicinity of a S one.
The six genes shown here are all differentially expressed in PL and S four of the genes have higher expression in pl, which does not have LIS one. Conversely, two have higher expression in S, which does have LIS one, We've just shown you how to grow inducible flax lines. So the genome changes in response to the growth environment and the growth characteristics of subsequent generations.
When doing this procedure, it's important to remember that the growth conditions need to be replicated for reproducible induction of changes. So that's it. I know many of you are skeptical of this phenomenon, but now you can reproduce the observations for yourselves.
Thanks for watching and good luck with your experiments.
