The oriental fruit fly is a highly invasive and adaptive pest species. Effective methods for functional gene research could help us to develop environmental-friendly pest management strategies. This method is high efficiency and low cost.
Our protocol will serve as a useful guide for generating mutant flies for oriental fruit fly researchers. To begin, predict the structure of target genes of interest and determine the boundaries between exons and introns via bioinformatic analysis of the Bactroceras dorsalis genome. Use the commercially available gRNA synthesis kit to generate the designed sgRNA.
Resuspend the gRNA product in nuclease-free water. Quantify the concentration using a UV-visible spectrophotometer and store at 80 degrees Celsius prior to use. Place B.dorsalis pupae into a plastic cage, with a rearing condition of 55%relative humidity, 26.5 degrees Celsius, and a 14:10 light/day cycle.
When the adults eclose, provide a mixture of sugar and yeast as food along with water. Most adults reach sexual maturity 10 days after emergence. Use a light stand with a light of 30 to 50 lux to improve the fecundity of females, therefore, improving the efficiency of embryo collection.
Next, place a 200-mesh gauze in the oviposition chamber, approximately 1 to 2 millimeters away from the chamber lid. Put a new oviposition chamber into the cage when the microinjection setup is ready. Collect the embryos every 10 minutes using a fine wet brush and line them up on a self-made injection plate.
Dip the embryos into halocarbon oil during injection to avoid further desiccation. Prepare the working solution by mixing the Cas9 protein and the corresponding sgRNA to a concentration of 300 nanograms per microliters of each. Then, add 1 microliter of phenol red to the mixture to serve as a convenient way to mark injected embryos.
Place the prepared mixture on ice to avoid degradation of the sgRNA. Prepare the glass injection needle using a micropipette puller. Add 3 microliters of the mixture into the injection needle without introducing air bubbles.
Then, open the needle using a Microgrinder and connect it to the micromanipulator according to the user guide. Set the parameters for the injector as Pi to 500 hectopascals. Ti to 0.5 seconds, and PC to 200 hectopascals.
Place the plate with lined-up embryos on the objective table. Then, adjust the micropipette position under a fine optical microscope, setting the micropipette and embryo on the same plane. Insert the needle tip into the posterior, or vegetal pole, of the embryo.
Then, press the pedal from the injector to deliver the mixtures into the embryo. The appearance of slight reddish color is due to the addition of phenol red in the mixture. Place the injection plate with the injected embryos into an artificial climate chamber.
After 36 hours, transfer the hatched larvae into larval diet using a fine-tip brush. Collect the larvae three or four times daily until no more larvae hatch. Next, place the mature larvae into the wet sand to pupate.
The pupation stage takes 10 days. After pupation, move the pupae to a plastic cage before eclosion begins. After isolating genomic DNA from a fresh puparium or a single mid-leg of individual adults and designing the specific primers to amplify the target area, perform polymerase chain reaction, or PCR, following cycling conditions described in the text manuscript.
Then, sequence the purified PCR products. Detecting multiple overlapping peaks adjacent to the target site suggests successful mutagenesis. For sub-cloning, mix the purified PCR products with a blunt-end vector and ligate them at 25 degrees Celsius for 15 minutes.
Then, add trans-T1 cells and place it on ice for 30 minutes. Then, add 500 microliters of LB medium and incubate at 37 degrees Celsius for 1 hour with shaking. Centrifuge and discard the supernatant.
Resuspend the pellet and spread it on an LB plate. Place the plate for overnight incubation at 37 degrees Celsius. Then, select individual bacterial colonies for sequencing to verify the genotype of the mutants.
In this study, the example of the target site of the selected gene is located in the third exon. The target site is highly conserved, and a single band was detected by gel electrophoresis for the DNA template for synthetic gRNA and gRNA obtained in vitro transcription. B.dorsalis survival and mutagenesis after microinjections are shown here.
After sequencing the PCR products, 80%of the G0 individuals are mosaic mutants. In mutant screening, the mutant with 8-base-pair deletion resulted in premature termination of amino acid translation. Inserting the needle into the posterior area or vegetal pole of an embryo is a central step because this directly affects the survival of eggs.
This technological provides a reference for studying areas of gene functions in B.Dorsalis They can quickly capture the mutants they want through this method.
