Osmia cornifrons vitellogenin: cDNA cloning, structural analysis and developmental expression

Abstract

Osmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. Osmia cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. Osmia cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C-terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus and 42%-30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachilidae, Apidae, Vespidae and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species. The expression profile of O. cornifrons vitellogenin mRNA during development revealed that O. cornifrons vitellogenin was first detected in the pupal stage and was continuously detected during the adult stage. Interestingly, O. cornifrons vitellogenin mRNA expression was low in mid-diapause, then gradually increased beginning on day 3 of the newly emerged adult stage, and subsequently declined. These results suggest that the expression level of O. cornifrons vitellogenin mRNA is stage-specific.

Ovarian development and secretion of vitellogenin protein during the wintering period and after emergence in the hornfaced bee, Osmia cornifrons

“…Additionally, the secretion level decreased slightly from the fresh enclosure of adults during month 1 of wintering, increased from month 1 to month 2, and then stagnated after 2 months. This result was similar to the development pattern of oocyte number and OcVg gene expression found in northern blotting during wintering (Lee et al, 2015). After diapause, OcVg protein was stage-specifically detected.…”

“…Clones harboring the cDNA insert were selected from the expressed sequence tags (ESTs) from a cDNA library that had been generated using entire O. cornifrons bodies (Lee et al, 2015). The plasmid DNA was extracted using the Wizard mini-preparation kit (Promega, Madison, WI, USA) and sequenced using an ABI 310 automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).…”

A serine protease inhibitor from the hornfaced bee, Osmia cornifrons, exhibits antimicrobial activities

“…The cDNA encoding OcSPI was isolated from expressed sequence tags (ESTs) generated using the O. cornifrons cDNA library (Lee et al, 2015). Plasmid DNA extraction was performed using a Wizard MiniPreparation kit (Promega, Madison, WI, USA) according to the manufacturer's protocols.…”

“…Ten O. cornifrons bees (Lee et al, 2015) were dissected after anesthesia by chilling and collecting tissue samples, such as the epidermis, fat body, gut, muscle, and venom gland. Tissue samples were washed three times with phosphate-buffered saline (PBS; 140 mM NaCl, 27 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4).…”

“…Total RNA was subjected to a 1.0% formaldehyde agarose gel electrophoresis (5 μg of total RNA/lane) and then transferred onto a nylon membrane (Schleicher & Schuell). Using the OcSPI cDNA as a probe, which was labeled with [α-32 P] dCTP (Amersham Biosciences) using a Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocols, hybridization was performed at 42°C in hybridization buffer (5 × saline-sodium citrate, 5 × Denhardt's solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA), as previously described (Lee et al, 2015). The membrane was washed with 0.1% SDS and 0.2× saline-sodium citrate at 65°C and then exposed to autoradiography film at −70°C.…”