Isolating of a putative glyceraldehyde-3 phosphate dehydrogenase (GAPDH) from Calophyllum brasiliense, an important tropical forest tree

Calophyllum brasiliense Cambess. has characteristics that made it an excellent candidate model for ecogenomics in rain forest trees such as widespread natural occurrence and geographical patterns of adaptive genetic variation. Besides, it is also becoming a popular species for reforestation efforts in Brazil. Although, very little is known about its genetic diversity and the molecular mechanisms involved genetic adaptation traits. The first difficulty in launching genetic studies in a wild wood species is the lack of an optimized protocol for RNA and DNA isolation. In this work we built the essential framework for molecular genetics research with C. brasiliense comparing four distinct methods of RNA extraction from of three different tissues: leaves, stems and roots. We also were successful in the isolation of genomic DNA by an optimized CTAB method. Finally, degenerated oligonucleotide primers were designed for isolating of the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene of C. brasiliense from the corresponding gene in closely related species. This gene is commonly used in plants as reference in expression gene analysis by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Primers for RT-PCR were designed based on partial sequence obtained using degenerate primers designed. The optimized methods of RNA and DNA extraction combined with the identification, isolation and specific primer design for RT-PCR of a traditional Reference Gene provide the essential framework for molecular genetics research with C. brasiliense.