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Molecular characterization of a stigma‐specific gene encoding an arabinogalactan‐protein (AGP) from Nicotiana alata
Author(s) -
Du He,
Simpson Richard J.,
Clarke Adrienne E.,
Bacic Antony
Publication year - 1996
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1996.09030313.x
Subject(s) - complementary dna , biology , nicotiana , microbiology and biotechnology , nicotiana tabacum , cdna library , arabinogalactan , signal peptide , gene , gynoecium , peptide sequence , biochemistry , botany , pollen , solanaceae , cell wall , stamen
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 Na 3) was isolated and sequenced. The AGP Na 3 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 pl of 7.5. Northern blot analyses showed that the AGP Na 3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension‐cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.