Localisation of Abundant and Organ-Specific Genes Expressed inRosa hybridaLeaves and Flower Buds by DirectIn SituRT-PCR

Abstract

In situPCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues.In situPCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants,in situRT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by usingin situRT-PCR inRosa hybridaflower buds and leaves. As target genes, we used the abundantβ-actinandRhFULgene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.