Shen J B, Hsu F C
E. I. DuPont de Nemours and Co., Wilmington, Delaware 19880-0402.
Mol Gen Genet. 1992 Sep;234(3):379-89. doi: 10.1007/BF00538697.
A cDNA library of Brassica napus (cv. Westar) was constructed using poly(A)+ RNA isolated from developing anthers of flower buds 2-3 mm in length. Differential hybridization, using cDNA probes complementary to poly(A)+ RNA from developing anthers or seedlings, was used for initial screening. In addition to Southern and Northern blot analyses of selected clones, RNA-PCR assays and in situ hybridization were used to study the temporal and spatial gene regulation in anthers at the transcriptional level. Five independent cDNA clones, showing no cross-hybridization to one another, were characterized, and their expression patterns could be grouped into three distinct categories. Two cDNA clones, BA112 and BA158, are tapetum-specific: the corresponding mRNAs accumulate in young anthers and decline as the tapetum cells degenerate later in anther development. The transcripts represented by BA54 and BA73 accumulate late in anther development and reach a maximum level in mature anthers prior to anthesis; BA54 has been confirmed to be pollen-specific. The third category, represented by BA42, is found to encode a protein sharing 64-67% amino acid similarity with chalcone synthase (CHS) from various plant species; the transcript is localized in the peripheral cells of the vascular bundle, tapetum, and developing microspores.
利用从长度为2 - 3毫米的花芽发育中的花药中分离得到的聚腺苷酸加尾RNA（poly(A)+ RNA）构建了甘蓝型油菜（品种：Westar）的cDNA文库。使用与发育中的花药或幼苗的聚腺苷酸加尾RNA互补的cDNA探针进行差异杂交，用于初步筛选。除了对选定克隆进行Southern和Northern印迹分析外，还使用RNA - PCR分析和原位杂交在转录水平研究花药中的时空基因调控。对五个相互之间无交叉杂交的独立cDNA克隆进行了表征，它们的表达模式可分为三个不同类别。两个cDNA克隆BA112和BA158是绒毡层特异性的：相应的mRNA在幼嫩花药中积累，并随着花药发育后期绒毡层细胞的退化而下降。由BA54和BA73代表的转录本在花药发育后期积累，并在开花前的成熟花药中达到最高水平；BA54已被证实是花粉特异性的。以BA42为代表的第三类被发现编码一种与来自各种植物物种的查尔酮合酶（CHS）具有64 - 67%氨基酸相似性的蛋白质；该转录本定位于维管束的外周细胞、绒毡层和发育中的小孢子中。
