Cloning And Expression Of AcOr170,AcOr10 And Selection Effect Of Drones On The Queen Mandibular Gland Pheromone In Chinese Honeybees,apis Cerana Cerana

Honeybee,a real social insect,has a complete and elaborate language communication system,and it is an important guarantee for the normal growth,development and reproduction of colonies and individuals.The queen mandibular gland pheromone is an important pheromone,which has important biological functions,such as regulating the workers behavior,promoting the workers to take part in collection activities and attracting drones during the queen mating flight.Based on the cloning and expression of the receptor gene 170(AcOr170)and the receptor gene 10(AcOr10)of queen mandibular gland pheromone in Chinese honeybees,Apis cerana cerana,this study explores the selection effect of drones on the queen mandibular gland pheromone.Taking Chinese honeybees(Apis cerana cerana)as the research object,a primer for the AcOr170 was designed to clone and analysis its cDNA sequence in the antennas of drones.The aim was to compare evolutionary differences between the Apis cerana cerana and other honeybee species.The experimental results showed that the overall sequence of AcOr170 was 1356 bp in length.The coding region length was 1188 bp,encoding 396 amino acids.Its molecular weight was 46.272 kDa,isoelectric point was 8.96,and the accession ID of Genbank was KX264359.Structural domain analysis indicated that the encoded protein contained a highly conserved structure of 7tm-6.Sequence alignment showed that genome sequences were highly similar among Apis cerana cerana,Apis mellifera linnaeus and Apis dorsata.Taking Chinese honeybees as the research object,the honeybee drone antennas were collected to extract total RNA and clone the sequence of Or10 using quantitative real-time polymerase chain reaction(qRT-PCR).The amino acid sequences of Or10 was analysed by bioinformatic software.The relative expression levels of Or10 in drone antennas and brains under unsexual maturity at hive entrance,sexual maturity at hive entrance,unsexual maturity at frames in hive,sexual maturity at frames in hive(respectively: group I,group II,group III,group IV)were detected using qRT-PCR.Results indicated that the cDNA sequence of Or10(named as AcOr10)(GenBank accession no.: MF693365)of Apis cerana cerana was obtained with length of 914 bp and coding region of 738 bp,which encodes 246 amino acids with molecular weight of 28.163 kD and isoelectric point of 5.50.The phylogenetic tree results showed that AcOr10(Apis cerana cerana)clustered with AmOr10(Apis mellifera)and AfOr4-like(Apis Florea).The qRT-PCR results of the expression of AcOr10 of drone antennas showed that the relative expression levels of AcOr10 in drone antennas in group II and group IV were significantly higher than that in the group I and group III(P<0.05),and group I and group III,group II and group IV were no significant differences in the expression levels of AcOr10 in antennas(P>0.05).The qRT-PCR results of the expression of AcOr10 of drone brains showed that the relative expression level of AcOr10 in brains in group II was significantly higher than that of group I,group III and group IV(P<0.05),while there was no significant difference among the latter three groups(P>0.05).Under the same physiological conditions,the relative expression levels of AcOr10 in antennas in group I,group III and group IV were all significantly higher than that in their brains(P<0.05),but there was no significant difference in the group II between antennas and brains(P>0.05).Taking Chinese honeybees as the research object.The drones were captured at hive entrance and frames in hive,were used to determine the attraction effect of three different queen mandibular gland pheromones on the drones using behavior observation method.The results showed that 10 ?g group was significantly higher than the 1 ?g and 100 ?g groups in the attraction of 9-ODA on the drones captured at hive entrance(P<0.05),which indicated that much higher or lower concentration of 9-ODA cannot effectively attract drones.The 1 ?g group has a higher difference than the 10 ?g and 100 ?g groups in the attraction of HOB on the drones captured at hive entrance(P<0.05),the effect illustrated that low concentration of HOB was more attractive to the drones captured at hive entrance.The 10 ?g and 100 ?g groups were significantly higher than the 1 ?g group in the attraction of HVA on the drones captured at hive entrance(P<0.05).Moreover,there were no statistical differences in the attraction of different concentrations of queen mandibular gland pheromones on the drones captured at frames in hive(P>0.05).