RESUMO

Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (-70 degrees C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.

Assuntos

Hevea/química , Látex/química , RNA de Plantas/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA de Plantas/genética , Eletroforese em Gel de Ágar , Biblioteca Gênica , Genes de Plantas , Hevea/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa