Molecular Cloning And Expression Of Chemosensory Protein Genes Family In The Chinese Honeybee. Apis Cerana Cerana(Hymenoptera: Apidae)

Chemoreception is a crucial biological process that is essential for thesurvival of insects, in which chemosensory proteins play a very important role. Apiscerana cerana is an indigenous, important economical bee species withcharacteristics such as olfactory sensitivity, cold resistance, heat hardiness, diseaseresistance and foraging efficiency, which is superior to western bees—Apis melliferaL. The objective of this study is to clone and analyze the whole chemosensoryprotein genes family in A. cerana cerana (Ac-CSPs), and study the biologicalfunction of Ac-CSPs by molecular biology methods. This may provide fundamentalevidence for the future study of the physiological function of CSPs.1. Six full-length of CSP genes of A. cerana cerana have been cloned by usingreverse transcription-polymerase chain reaction (RT-PCR). The GenBank accessionof the five gene (Ac-CSP1, Ac-CSP2, Ac-CSP4, Ac-CSP5and Ac-CSP6) isFJ157352、FJ157353、JQ979432、JQ979433and JQ979434. Five full-length ofAc-CSP genes were cloned in which Ac-CSP1was351bp in full length that encodeda polypeptides containing116amino acids with prediction of MW (molecular weight)of13.85kD and pI (isoelectric point) of4.89, respectively. Ac-CSP2was354bp infull length that encoded a polypeptides containing117amino acids with prediction ofMW of13.01kD and pI of8.86, respectively. Ac-CSP4was387bp in full length thatencoded a polypeptides containing14.61amino acids with prediction of MW of14.61kD and pI of4.53, respectively. Ac-CSP5was315bp in full length thatencoded a polypeptides containing104amino acids with prediction of MW of12.46kD and pI of9.60, respectively. Ac-CSP6was378bp in full length that encoded apolypeptides containing125amino acids with prediction of MW of14.63kD and pIof8.71, respectively of all with four conserved cysteine residues. The six deducedAc-CSPs sequences showed high sequence similarity (95%~99%) as compared withCSPs sequences of A. mellifera.2. The expression patterns at development phases and in different sensoryorgans of the six Ac-CSPs were analyzed by real-time PCR. The genes of Ac-CSP1, 2,3,4were not expressed in eggs, larvae and pupae, with the Ac-CSP1,2,4expressed at highest level in antennae and Ac-CSP3was stronger expressed in wings.The expression of Ac-CSP5was not significant difference at all developmental stages,while Ac-CSP6only highly expressed in the pupae. The expression patterns ofAc-CSPs and Am-CSPs are similar but there is a part of difference in this research.3. The ORF sequence of Ac-CSP1, Ac-CSP2and Ac-CSP4were subcloned intoprokaryotic vector and all expressed with the form of solubility. Then the Ac-CSPswere purified by Ni2+-NTA agarose gel.4. The competitive fluorescence assay was used to determine the bindingfunction of Ac-CSP1, Ac-CSP2and Ac-CSP4with odors having different structures.In the competitive fluorescence assay between Ac-CSP1, Ac-CSP2and Ac-CSP4with N-Phenyl-1-naphthylamine (1-NPN), the dissociation constants K1-NPNofAc-CSP1, Ac-CSP2and Ac-CSP4were1.24μmol/L,1.25μmol/L and2.99μmol/L,respectively. In the competitive binding assays， seven candidate ligands reduced therelative fluorescence intensity of1-NPN by more than50%，with β-ionone causing90%reduction in the relative fluorescence intensity；With the result that the genes ofAc-CSP1,2,4were not expressed in eggs, larvae and pupae indicating that Ac-CSP1,2,4may act as a carrier forodor molecules released from flower and nectar and playa role in searching for honey sources.