IDENTIFICATION AND IN-SITU LOCALIZATION OF POLLEN-SPECIFIC GENES

This chapter examines the ways in which the expression of haploid genes and their products can be localized. One way of identifying genes that are transcriptionally active in a particular cell is to examine the mRNA population present in that cell. By isolating poly (A)RNA from pollen or anthers, cDNA libraries can be successfully constructed in which each unique cDNA clone represents the transcript of a gene that is active in these tissues. The identification of cDNA clones corresponding to mRNAs that are uniquely transcribed in pollen or anthers can then be done by a process of differential hybridization. Total RNA is isolated from pollen or anthers along with RNA from a number of sporophytic tissues, such as stems, roots, and leaves. Pollen development is correlated with a number of morphological markers—including floral bud length, hypanthium length, or anther length—and such markers are used for describing the various stages of microsporogenesis. The temporal expression patterns of pollen- or anther-specific genes can, therefore, be determined by isolating RNA from flower buds of different lengths to identify when the gene transcript first appears. This can then be related back to a particular stage of pollen development. © 1992, Academic Press Inc.