RNAi has become an effective method for identifying the function of genes in nematodes and are proposed as a new way to effectively control pathogenic nematodes. RNAi is soaking nematodes in dsRNA can suffice for the procedure. To begin, collect the nematodes by the Baermann funnel method.
For this, place a clamped rubber tube below a funnel and place two layers of filter paper in the mouth of the funnel. Transfer the Botrytis cinerea fungal cultures to the funnel and add water to immerse the fungal mat. When nematodes are collected, add 500 microliters of extraction reagent and 100 microliters of magnetic beads to a two milliliter centrifuge tube.
Aspirate 20 microliters of the nematodes and grind the samples at 9, 000 x g for 30 seconds. Incubate for five minutes and centrifuge at 12, 000 x g for 10 minutes at four degrees Celsius. Transfer the supernatant to a fresh centrifuge tube.
Add 100 microliters of chloroform and mix by inverting the tube several times. Incubate for three minutes and then centrifuge at 12, 000 x g for 10 minutes at four degrees Celsius. Again, transfer the supernatant to a fresh centrifuge tube.
Add 250 microliters of isopropyl alcohol and vortex vigorously. Centrifuge at 12, 000 x g for 10 minutes and vortex. Discard the supernatant and add 500 microliters of 75%ethanol to wash the RNA.
Vortex the sample followed by centrifugation at 12, 000 x g for five minutes at four degrees Celsius. Air dry the RNA pellet for five minutes and resuspend it in 30 microliters of RNase free water. Calculate the RNA concentration using this formula and calculate the A260 to A280 ratio.
Next, use a pair of primers to amplify the partial coding sequence of the BX ppm-1 gene from the B.xylophilus. Then clone the ppm-1 one gene sequences into a pGEM-T Easy vector containing the T7 promoter. Recover the 894 base pair long ppm-1 gene fragment by using the cloned plasmid as the template for pcr.
Then, use an in vitro transcription kit to synthesize the dsRNA. Analyze the quality of the dsRNA using a spectrophotometer and visualize the products on a 1%agarose gel. Add four microliters of 5X soaking buffer and make up the volume to 20 microliters with double distilled water to get a final RNA concentration of 0.8 micrograms per milliliter.
Then place the nematodes in the dish for 30 minutes and wait for the eggs to adhere to the bottom. Carefully remove the water and nematodes without disturbing the eggs until only the eggs remain in the dish. Hatch the collected eggs in the dark at 25 degree Celsius for 24 hours to obtain J2 larvae.
Collect the larvae in a tube and wash them three times with double distilled water. Then, transfer the larvae into the tube containing the dsRNA solution. To this, add resorcinol solution to get a 1%solution.
Place the larvae on a shaking table to ensure sufficient absorption of dsRNA into the larvae. For controls, soak the same quantity of nematodes in the soaking buffer without the dsRNA probe or with a GFP dsRNA probe. Perform a qPCR using the actB and tbb-2 genes as internal references to evaluate the changes in gene expression level.
Use the delta delta Ct method to estimate the relative gene expression level from the dissolution curve and Ct value. After RNAi, culture the J2 larvae until adulthood on Botrytis cinerea lawns on PDA plates. Collect the adults using the Baermann funnel method as shown in section one.
Acquire images of the adult nematodes under a microscope and use the ImageJ software to measure the body length of 50 male and 50 female nematodes. Analyze the data by calculating the mean and standard deviation for each sample. Apply Student's t test to compare the means of the samples from the different groups.
Relative gene expression analysis after RNAi indicated that the ppm-1 dsRNA can effectively inhibit the expression of the ppm-1 one gene of B.Xylophilus. Whereas exogenous GFP dsRNA had no effect on the ppm-1 expression. After RNAi, the size of the adults markedly decreased despite attaining sexual maturity resulting in the small body size mutant phenotype.
The mean body length of the mutant females and males was 544 and 526 micrometers compared to 971 and 912 micrometers for the control group The most important thing is to transfer the nematodes into the double strand RNA solution and add another reagent to stimulate the nematodes to feed. This method is suitable for large scale gene screen and is commonly used in cell research. Studying the gene function of B.Xylophilus by this method has guiding value for the biological control of B.Xylophilus.
