You want to discover the role of a gene (named ABC) of a fungal pathogen that is predicted to be involved in infection of canola. There is little sequence information for this fungus, but the sequence of ABC is available. This gene has a single intron (60 bp in length). When the wild type fungus is grown on complete medium, after 5 days gene ABC is transcribed to a level of 0.5% to that of actin from the pathogen. You have designed a RNAi gene construct to silence ABC and have transformed the construct into the wild type fungal isolate. You need to screen transformants and select an isolate in which transcription of ABC is reduced to less than 5% of that of the wild type when grown in culture for 5 days. Describe approaches you would use to identify appropriately-silenced fungal transformants and outline the controls you would include.
The aim of the experiment is to
generate a transgenic which does not express the ABC gene.
For this experiment, an antisense sequence is cloned into the
fungus. The transgenic confirmation can be performed by several
methods.
i. Northern blotting:
Isolate RNA from the WT, RNAi-transgenic, and a known mutant for
ABC gene (If available)
If the RNAi is functional, we would not detect any band in the RNAi
transgenic.
WT = Positive control
Mutant = Negative control
ii. Real-time PCR:
Isolate RNA from the WT, RNAi-transgenic, and a known mutant for
ABC gene (If available). Convert it to cDNA and perform real-time
PCR.
If the RNAi is functional, we would not detect any PCR band in the
RNAi transgenic.
WT = Positive control
Mutant = Negative control
You want to discover the role of a gene (named ABC) of a fungal pathogen that...
2. A dominant allele H reduces the number of body bristles that Drosophila flies have, giving rise to a “hairless” phenotype. In the homozygous condition, H is lethal. An independently assorting dominant allele S has no effect on bristle number except in the presence of H, in which case a single dose of S suppresses the hairless phenotype, thus restoring the "hairy" phenotype. However, S also is lethal in the homozygous (S/S) condition. What ratio of hairy to hairless flies...