Du H, Simpson R J, Clarke A E, Bacic A
School of Botany, University of Melbourne, Parkville, Victoria, Australia.
Plant J. 1996 Mar;9(3):313-23. doi: 10.1046/j.1365-313x.1996.09030313.x.
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 (AGPNa3) was isolated and sequenced. The AGPNa3 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 AGPNa3 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.
从烟草的雌蕊中分离出阿拉伯半乳聚糖蛋白（AGPs），进行去糖基化处理，然后按照之前报道的方法，通过反相高效液相色谱法对蛋白骨架进行分级分离。分离出一个主要级分RT35，经蛋白酶消化后获得肽序列。根据氨基酸序列设计了一个基因特异性简并寡核苷酸，并从雌蕊RNA中体外扩增出一个380 bp的PCR片段。该PCR片段用于筛选雌蕊cDNA文库，分离并测序得到一个762 bp的cDNA克隆（AGPNa3）。AGPNa3 cDNA编码一个169个氨基酸的蛋白质，该蛋白质由三个结构域组成：一个N端分泌信号、一个富含脯氨酸的结构域和一个C端富含半胱氨酸的结构域。成熟蛋白有145个氨基酸残基（16.7 kDa），预测的等电点为7.5。Northern印迹分析表明，AGPNa3基因仅在烟草及其近缘烟草属物种的雌蕊中表达，而在其他植物或悬浮培养细胞中不表达。进一步的Northern印迹分析和原位杂交表明，在雌蕊中，它主要在成熟花的柱头组织中表达。
