Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein (AGP) from Nicotiana alata

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata, deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA, The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGP Na3) was isolated and sequenced. The AGP Na3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pi of 7.5. Northern blot analyses showed that the AGP Na3 gene was only expressed in the pistils of IV. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in site hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.