Summary
In the
past two decades, chloroplast genetic engineering has been advanced to achieve
high-level protein accumulation but not for down-regulation of targeted genes.
Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450
monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast
genome to study RNA interference (RNAi) of target genes in intended hosts. PCR
and Southern blot analysis confirmed homoplasmy and site-specific integration
of transgene cassettes into the chloroplast genomes. Northern blots and
real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA
transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the
abundance of cleaved dsRNA was greater than the endogenous psbA transcript.
Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased
transcription of the targeted gene to almost undetectable levels in the insect
midgut, likely after further processing of dsRNA in their gut. Consequently,
the net weight of larvae, growth and pupation rates were significantly reduced
by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs
via the chloroplast genome for the first time opens the door to study RNA
interference/processing within plastids. Most importantly, dsRNA expressed in
chloroplasts can be utilized for gene inactivation to confer desired agronomic
traits or for various biomedical applications, including down-regulation of
dysfunctional genes in cancer or autoimmune disorders, after oral delivery of
dsRNA bioencapsulated within plant cells.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522700/?report=classic