The Laboratório de Genética de Abelhas (Dep. Biologia, FFCLRPUSP) is actively involved in the sequencing and annotation of the honey bee genome. Nucleotide sequences of this insect of great economic importance are now available (Amel 3.0,http://www.hgsc.bcm.tmc.edu/projects/honeybee/), and has greatly opened the opportunity to study the function and regulation of genes of interest using honey bee as a model-animal. In this direction, the integument (formed by epidermis and its secretion product, i.e., the exoskeleton) is an excellent tissue for studies of genes that are critical for the success of arthropod morphogenesis. This tissue, which is the target of complexes modifications attributed to the fluctuation of ecdysteroid hormones, expresses a gene (Amlac2) which encodes a laccase enzyme with a putative function in exoskeleton differentiation. In our previous study (FAPESP, proc. 04/09614-2), the Amlac2 cDNA was characterized in honey bee integument. The transcriptional pattern showed correspondence with the integument differentiation, as well as with ecdysteroid titer fluctuation. The aim of the present project is to study the function and regulation of Amlac2 gene. We intend to (1) characterize the Amlac2 structure, (2) analyze (qualitatively and quantitatively) Amlac2 expression during critical periods of ontogenesis and in relation to integument differentiation, (3) quantify endogenous ecdysteroid titer and perform experiments of hormone manipulation, aiming to verify whether the function of Amlac2 is hormonally regulated, (4) use interference RNA (RNAi) for Amlac2 silencing. In this case, possible changes in the exoskeleton structure will be investigated using microscopy methods. This comprehending approach certainly will permit to characterize both, the function and regulation of Amlac2 in the context of development.
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