The overall goal of this procedure is to develop an agro bacteria mediated virus induced gene silencing assay in cotton to study gene function. This is accomplished by first growing cotton seedlings to the 10 days to two week old stage. Then the cotton chloroplasts rados one gene is cloned into a tobacco rattle virus based vs vector.
The third step is to prepare agrobacterium cultures containing P-T-R-V-R-N-A one and P-T-R-V-G-R-C-A one. The final step is to hand inoculate the agrobacterium cultures into cotton cotter leadin. Ultimately, results can be shown for gene silencing by observing the albino phenotype on the true leaves of cotton plants about two weeks later.
The main advantage of this technique over existing methods like transformation is that VIGs provides a rapid and efficient platform for gene function study in cotton. Though this method can prove to be a powerful tool for rapid large scale analysis of gene function at the genome wide level in cotton, it can also be applied to other important crop species. Generally, individuals new to this method will struggle because it is difficult to hand inoculate the agrobacterium cultures and cotton coan.
This method can help answer key questions in the cotton genetics and genomics field, such as exploring gene functions To grow cotton seedlings for virus induced gene silencing. Add metro mix 700 to several seven centimeter pots and place the filled pots into a tray. Next plant the seeds of several upland cotton varieties such as fiber max 8 3 2 phyto, gen 4, 2 5, RF phyto, gen four 80 WR, and delta pine 90.
In the prepared pots, cover the tray containing the pot with a plastic dome and place the tray into a growth room, a 23 degrees Celsius, 120 micro Einsteins per square meter per second light with a 12 hour light, 12 hour dark photo period after three to four days, once two olean have emerged, remove the plastic dome. Allow the cotton plants to grow for around 10 days until the plants reach the stage where two fully expanded cot leadin have developed. But before true leaves have emerged, the plants are then ready to be used for the virus induced gene silencing assay.
These cloning procedures should be performed during the two week period when the cotton plants are reaching the fully expanded stage. First, amplify the cotton chloroplasts one gene from a CD NA library of CACI remondi leaf tissues using the primers provided in the written protocol according to standard PCR procedures. Then digest the GCA one PCR product with Echo R one and K PN one according to the enzyme manufacturer's protocols.
Visualize the 500 base pair PCR product on an Athene bromide gel. Cut out the band and extract the DNA ligate, the purified PCR product into the PYL 1 56 vector. Using T four DNA Ligase transform competent e coli cells with the ligated vector and then spread 10 to 50 microliters of the transformed cells onto LBA agar plates containing 50 micrograms per milliliter of canam mycin.
Incubate the plate overnight at 37 degrees Celsius the next day. Pick colonies from the plate and grow overnight in two milliliters of LB broth containing 50 micrograms per milliliter of can mycin harvest the bacteria by centrifugation and perform DNA preps according to standard procedures. To purify the plasmid DNA, verify the constructs by restriction enzyme digest and sequencing.
Once the presence and orientation of the insert in the PYL 1 56 vector is confirmed, electro operate the plasmid into agrobacterium tumor fas GV 3 1 0 1 and recover in LB liquid medium at 28 degrees Celsius. Select the transformants on LBI agar plates containing 50 micrograms per milliliter of can mycin and 25 micrograms per milliliter of gentamycin. If desired, the bacteria can be stored in 25%glycerol at minus 80 degrees Celsius.
Three days before inoculation streak T thought glycerol stocks of agrobacterium tumor facies carrying PYL 1 9 2 RNA one PY, 1 56 RNA two and PY 1 56 RNA two GLA one on LB antibiotic agar plates incubate the plates at 28 degrees Celsius for 24 hours. The next day, pick a single colony for each construct and inoculate each colony into five milliliters of LB medium. Supplemented with 50 micrograms per milliliter of can mycin and 25 micrograms per milliliter of Gentamycin.
Grow the bacterial culture at 28 degrees Celsius overnight in a shaker set at a speed of 50 rotations per minute. Transfer the PYL 1 56 RNA two GLA one culture and control cultures to separate flasks containing 50 milliliters of LB medium supplemented with 50 micrograms per milliliter of can mycin and 25 micrograms per milliliter of Gentamycin plus 10 millimolar MES and 20 micromolar aceto. Cy Ringy Grow the culture at 28 degrees Celsius overnight in a shaker set at a speed of 50 rotations per minute.
The following day, transfer the cultures to centrifuge tubes and spin down the agro bacterial cells at 4, 000 rotations per minute For five minutes, re suspend the culture in the infiltration buffer containing 10 millimolar magnesium chloride, 10 millimolar MES and 200 micromolar aceta Tyrone. Measure the OD 600 of the culture and adjust to 1.5 with infiltration buffer. Incubate the culture on the bench at room temperature for three hours prior to agro bacterial leaf infiltration.
Punch the underside of cott leadin of cotton plants with a 25 gauge needle without piercing through the cott leadin. One or two holes are punched on each section of Cott Leadin. Next mix the PYL 1 92 RNA one agro bacterial culture with either PYL 1 56 RNA two or PY 1 56 RNA two GRCA one in a one-to-one ratio.
Then using a one milliliter needle or syringe. Introduce the mixture into the underside of the cott lead-ins through the punched holes. Cover the plants with a plastic dome and leave the infiltrated plants at room temperature under dim light conditions overnight.
Transfer the plants to a growth room with a temperature of 23 degrees Celsius, 120 micro Einstein per square meter per second light with a 12 hour light 12 hour dark cycle. Examine the silencing phenotype at seven to eight days postle infiltration. At this point, the true leaves of plants silenced by PY 1 56 RNA two GLA one begin to display the albino phenotype.
The plants infiltrated with agro bacteria carrying PYL 1 56 RNA two. Serve as a control. Verify the gene silencing efficiency by examining the expression level of endogenous genes using reverse transcription PCR with RNA, isolated from control and silenced cotton plants according to standard procedures.
This image shows a fiber max 8 3 2 cultivar after virus induced gene silencing of the GLA one gene. Note the white colored leaves where gene silencing has occurred here. A PHYTO four 80 WR cultivar also exhibits the albino phenotype after virus induced gene silencing.
Similarly, this phyto 4 2 5 RF cultivar displays white leaves where virus induced gene silencing has occurred. Finally, this image shows another successful viral induced gene silencing experiment this time in Delta PINE 90 cultivars. While attempting this procedure, it's important to remember to add acetone into the agrobacterium cultures before leaving them on the bench for the three hours following this procedure.
Other methods like vig, CD NA library cloning can then be performed in order to answer additional questions like exploring novel gene functions after its development. This technique paved the way for researchers in the field of plant genetics to explore the gene function and defense response to antibiotic an biotic stresses in economically important crop Plants. Once mask data, this technique can be done in two to three days if it's performed properly.
After watching this video, you should have a good understanding of how to perform virus induced gene silencing in cotton.
