Isolation and characterisation of two pollen-specific LIM domain protein cDNAs from Nicotiana tabacum

Abstract 

A PCR-generated DNA probe covering the region corresponding to the two contiguous LIM domain sequences from the sunflower PLIM-1 cDNA was used to screen a cDNA library prepared from poly-A⁺ RNA isolated from mature Nicotiana tabacum pollen. Two clones were isolated and found to have unique but closely related nucleotide sequences NTPLIM1a and NTPLIM1b. Homology searches revealed the presence of two LIM domain repeats in each clone. Detailed spatial and temporal northern blot analysis showed that the NTPLIM transcripts were present specifically in mature and germinating pollen and were detectable immediately before the asymmetrical division pollen mitosis I, reaching a maximal level in mature pollen. Southern blot analysis revealed the NTPLIM1a and NTPLIM1b genes to be single-copy and members of a small gene family in the N. tabacum genome. Analysis of a genomic clone encoding NTPLIM1a revealed that the gene is split by four introns ranging from 79 to 922 bp in length. The high levels of developmentally regulated expression specifically in pollen suggests an important role for these proteins in pollen maturation and/or function.