Construction Of The Head ESTs In Apis Cerana Cerana, And Its Main Antennal Proteins: Molecular Cloning, Immunocytochemical Localization And Expression

Apis cerana cerana is an indigenous, important economical bee species with about two million colonies currently maintained in China. The olfaction of A. cerana cerana seems more exquisite than that of western bees—Apis mellifera L, which provides more powerful ability of making good use of the odd honey resource. Odorant binding proteins (OBPs) may directly participate in the recognizing progress of olfactory sensilla to the environmental odors. In addition, A. cerana cerana has the characters of subtle distinguishing ability and intensive discriminate against others, and the adaptive resistance to its natural parasitic mites—Varroa jacobsoni, in such progress, chemosensory proteins (CSPs) may participate in the action of discrimination against others and resistance to mites.In the dissertation, a cDNA library from heads of A. cerana cerana was constructed and functional genes from hundreds of expressed sequence tags (ESTs) by patched sequencing were discovered from cDNA library. On the basis of ESTs, three antenna specific protein (ASP)—Ac-ASP1, Ac-ASP2 (both belong to OBPs) and Ac-ASP3 (belongs to CSPs), were cloned and identified through the transcriptional profiles and immunocytochemical localization in antennal sensilla. All above will produce an important basis to the exquisite olfaction and action of discrimination against others and resistance to mites of A. cerana cerana. The results showed that:1. The construction of cDNA library with 13 days' A. cerana cerana. Through the technique of LD-PCR, ds-cDNA was synthesized and low abundance mRNA was also amplified with high limit. There were several clear bands, which might be some high abundance genes, with molecular length 0.6～1.0 kb in the electrophoretic results. The cDNA library was identified high recombinational ratio (reached above 90% and 97.6%, respectively) through identification of PCR and blue-white selection, and having high capability of clones (the titer of unamplified library reached 1.25×10⁶ pfu/ml and amplied library reached 1.74×10⁹ pfu/ml);2. Based on the cDNA library, 862 ESTs were obtained through batched sequencing, and 68 "Contig" and 184 "Singlton" were produced from the assembled ESTs. After sequence annotation some high expression abundance genes, including major royal jelly protein, MRJP1; major royal jelly protein, MRJP2, glucose oxidase, GOX and Apisimin etc, and including odorant-binding protein 15 related to information transmission in antenna, were found and such functional genes provided foundation for next research;3. Based on information of A. mellifera, three important genes encoding antenna specific proteins, ASP: Ac-ASP1, Ac-ASF2 and Ac-ASP3, were cloned. All of them had been submitted into GenBank and the registered IDs are DQ449670, DQ44966 and DQ449669, respectively. From the nuclear acid sequences, Ac-ASP1 and Ac-ASP2 have 6 conservative cysteines and Ac-ASP3 has 4 ones. After analysis of multiple sequences alignment, phylogenetic tree and similarity of sequences, it was shown that Ac-ASP has similar relation with Am-ASP, and shared high homology with other Hymenopteran and Coleopteran. From above analysis, we firmly suggest Ac-ASP1 and Ac-ASP2 belong to OBPs family and Ac-ASP3 belong to CSPs family.4. Temporal and spatial expression profile of the three genes: ac-ASP1, Ac-ASP2 and Ac-ASP3 were analyzed by real-time PCR: Ac-ASP1, expressed higher in antenna, but less in other tissues including head, thorax, abdomen, wings and legs; Ac-ASP2 only expressed in antenna but never found in other tissues, Ac-ASP3 widely expressed in all tissues inhomogeneously. In the different developmental period, Ac-ASP\ expressed in two highest and quite abundance periods: from larva to 6-day adult and around 21-day adult; Ac-ASP2 did not express in larva and pupa, but have two discontinuous high abundance periods: 9, 27-day and 1, 15, 30-day, the expressing abundance at the two periods were tenfold higher than those at other periods; Ac-ASP3 also have two high expressed periods on 6-day and 18-day, and the expressing abundance at 6-day was about double as 18-day.5. The ORF sequence of Ac-ASP2 and Ac-ASP3 were subcloned into prokaryotic vector and expressed with the form of inclusion and solubility, respectively. The antigen yield to Ac-ASP2 can reach 50 mg per liter LB medium after purified by washing and dissolving of urea accumulated up, and the antigen yield to Ac-ASP3 can reach 15 mg per liter LB medium after purified by Ni²⁺-NTA agarose gel. Ac-ASP3 is better than Ac-ASP2 from the purifying effect but worse than that from the yield ratio. As antigen the two recombination proteins of Ac-ASP2 and Ac-ASP3 were prepared for polyclonal antisea, whose potency can reach 1:32 and 1:64 respectively through double immunodiffusion tests. Both of them reached the requirement for preparation of immune probes.6. The immune colloid probes of Ac-ASP2 and Ac-ASP3 were prepared, the distribution pattern in antenna sensilla subcellular localization of them were observed through Transmission Electron Microscope (TEM). From the results, Ac-ASP2 was deduced to participate in the olfactory recognization process according that mainly expressing on olfactory sensilla: sensilla placodea (sp) and sensilla trichoidea A (stA); Ac-ASP3 was presumed to play a role in the mechanical process for expressing on mechanical sensilla: sensilla basiconica (sb) and sensilla trichoidea B (stB). All of above will provide for important reference to the functional mechanism of olfactory and chemosensory system in A. cerana cerana.