Amblyomma americanum salivary glands: double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition of PGE2 stimulated protein secretion.
Protein secretion into the saliva from the tick salivary glands is due to exocytosis of vesicular membrane bound granular material regulated by SNARE complex proteins after salivary gland stimulation by PGE2 [Insect Biochem. Mol. Biol. 32 (2002) 1711]. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. Synaptobrevin is a key v-SNARE in all secreting cells studied to date. A vesicle-associated synaptobrevin cDNA fragment homologue from the salivary glands of partially fed lone star tick females was cloned and sequenced. Double-stranded (ds) RNA interference (RNAi) is an effective method to silence specific gene expression. The functional role of synaptobrevin in protein secretion in partially fed tick salivary glands was studied with an in vitro RNAi method. Incubation of isolated salivary glands with double-stranded RNA (dsRNA) transcribed from a tick salivary gland synaptobrevin cDNA fragment resulted in decreased expression of the transcript, a reduction in the level of synaptobrevin protein and inhibition of PGE2 stimulated anticoagulant protein secretion by isolated salivary glands. We demonstrate the applicability of RNAi for studying individual steps in the mechanism of PGE2 stimulated exocytosis in the salivary glands of ixodid ticks.
Karim
Shahid
S
Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK 74078-3033, USA.
Ramakrishnan
Vijay G
VG
Tucker
James S
JS
Essenberg
Richard C
RC
Sauer
John R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D015232:Dinoprostone / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D005089:Exocytosis; D020868:Gene Silencing; D026863:Ixodidae / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D050683:R-SNARE Proteins; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012469:Salivary Glands / Q000187:drug effects / Q000378:metabolism*; D016415:Sequence Alignment; D014158:Transcription, Genetic
D001779:Blood Coagulation Factors; D018076:DNA, Complementary; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D015232:Dinoprostone