The main difference between queens and workers of Apis mellifera is the size of their ovaries. Whereas in queens, each ovary is composed of 150-200 units serial units, termed ovarioles, this number is much lower in workers, ranging from only 2-20. This contrast is the anatomical basis of reproductive division of labor and caste function. The differences between the two ovarian phenotypes are established in the third to fifth larval instare, when most of the ovariole primordia undergo autophagic cell death in larvae that develop into workers. Analysis of differential gene expression in ovaries of fifth instar queen and worker larvae revealed the over-expression of a considerable number of genes of unknown function and even unpredicted ones. One of the unpredicted genes was validated as over-expressed in the worker ovary. It is located in the genome scaffold Group 11.31and is predicted to encode a long non-coding RNA, preliminarily named long-non-coding-ovary-1 (lncovary-1). The full length cDNA is 1368 bp long and located within an intron of the predicted protein-coding gene LOC726407, for which no function is known. Besides being the first non-coding RNA (lncRNA) discovered in Apis mellifera, it is of particular interest as it maps to a central position within a QTL for variation in ovariole number in honey bee workers. The current project aims to functionally characterize the lncovary-1 gene, as well as the one within which it is located (LOC726407). The first step will be to analyze the expression patterns of these genes during ovary development of worker larvae by means of real time RT-PCR. The second objective is to further investigate the intracellular localization of the lncovary-1 RNA. Through FISH analysis it was detected as concentrated in a defined spot in the proximity of the nucleus in ovaries of honey bee larvae, which leads us to believe that this lncRNA may be involved in specific processes of cell differentiation. In such experiments we will combine lncovary-1FISH analysis with organelle-specific markers. Finally, having obtained data on the expression of the two genes, will try to carry out experiments of their function through RNAi-mediated knockdown. RNAi experiments in honeybees are already established for several genes, but non has involved so far lncRNAs.
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