Molecular Cloning,Spatiotemporal Expression And Functional Analyses Of The Takeout Genes In Apis Cerana Cerana And Apis Mellifera Ligustica

Takeout(to)is a circadian clock-regulated output gene abundantly expressed in several tissues related to perception of chemical signals and nutrition acquisition in insects.TO protein is known to be involved in physiological metabolism,growth and development,and behaviors regulation in solitary insects.But it remains unclear whether TO protein is also involved in the division of labor in social insects.In order to address this general question,we cloned the to genes from two social insects,Apis cerana cerana and Apis mellifera ligustica,and analyzed their expression patterns in different developmental stages(nurse bees vs.forager bees)and different tissues of the two species.Furthermore,We also knocked down to gene to test if it is involved in regulation of labor division in Apis mellifera ligustica by using RNAi(RNA interference)technology.The results obtained are summarized as follows:The cDNA sequences of to gene in Apis cerana cerana(named AcTO1,1058 bp)and Apis mellifera ligustica(named AmTO1,783 bp)were cloned by RT-PCR.Sequence analysis showed that the ORFs of the two genes were both 738 bp in length,encoding a protein of 246 amino acids.The molecular weights of the translated proteins of AcTO1 and AmTO1 are 27.09 and 27.07 kDa,respectively.The two TO proteins had an identical isoelectric point(PI)of pH 8.40.Both AcTO1 and AmTO1 contained a N-terminal signal peptide of 17 animo acid residues but lack transmembrane domains,which suggested that they were secreted proteins.The two TO proteins also shared an highly conserved JHBP(juvenile hormone binding protein)domain,the signature domain of the JHBP superfamily genes in insects.We monitored the expression patterms of AcTO1 and AmTOl(head,thorax and abdomen)of 3 types of foragers(OF1 and 2:Age-matched/old foragers 1 and 2;YF:Young/precocious foragers)and 2 types of nurses(YN:Age-matched/young nurses;ON:Old/over-aged nurses)by using Real-time quantitative PCR(qRT-PCR).In the 3 types of foragers and 2 types of nurses of the two species,to expressed at the highest level in the head,followed by in the thorax,and then in the abdomen.Moreover,AcTO1 and AmTO1 transcripts in the head were significantly higher in the 3 types of forager bees than in the 2 types of nurse bees in both species.Such expression profiles of the TO gene in both bee species implied that TO was probably associated with the division of labor between nurses and foragers.To further test if to indeed was involved in the division of labor in the honeybee,we fed 16-day-old worker bees of Apis mellifera ligustica with AmTO1 dsRNA,DEPC H2O(control),or DsRed(dsRNA control)dsRNA.Quantitative RT-PCR analyses showed that workers fed with AmTO1 dsRNA had significantly lower levels of AmTO1 transcripts in the head than those fed with DsRed dsRNA or DEPC H2O controls.Moreover,the number of workers fed with AmTO1 dsRNA that went out and collected pollens/nectars was significantly fewer than those fed with DsRed dsRNA or DEPC H2O controls.These results directly demonstrated that AmTO1 was required for foragers to perform their normal task-foraging behavior.