Laser Microdissection-Next Generation Sequence Transcriptome Analysis in the Papilla Cell of Brassica rapa

Flowering plants have several mechanisms in pollination and fertilization. Pollonation is performed through interaction of pollen and pistil, and its process begins when pollen grain attaches to papilla cell of a stigma in pistil. Recent studies have revealed that various factors are involved in pollen–pistil interactions, but these mechanisms are still unclear in detail. To understand and molecular basis of pollination and fertilization, we focused on a transcriptome analysis for the papilla cells of Brassica. rapa by a combination of Laser Microdissection (LM) and Next generation sequencing (NGS). We investigated a suitable fixative for papilla cells. Floral buds before anthesis were fixed in following fixative conditions: 75% ethanol/25% acetate, 60% ethanol/40% acetate, 100% ethanol and 100% acetone. Fixed buds were embedded in paraffin by microwave method, and papilla cell specific cell types were isolated from cross sections of flower bud by LM. Total RNAs were extracted from LM cells and were applied to NGS analysis using SOLiD4 (Applied Biosystems ). The quality of Total RNA was the best from papilla cells fixed in 100% acetate, and the RNA Integrity Number (RIN) was more than 6.0. We are now profiling gene expression in papilla cells using the B. rapa genome sequence. The acquired gene profiling will clarify the chromosomal position of the genes which function in the papilla cells, and predict the genes which participates in pollen-pistil interaction.