Maucher H P, Scheuerlein R, Schraudolf H
Institut für Allgemeine Botanik, Universität Ulm, Albert-Einstein-Allee 11, Germany.
Photochem Photobiol. 1992 Nov;56(5):759-63. doi: 10.1111/j.1751-1097.1992.tb02231.x.
Phytochrome controls several developmental steps during formation and differentiation of the fern gametophyte, including spore germination, morphogenesis of the gametophyte or differentiation of the sexual cells. To obtain information about the amino acid sequence and the regulation of phytochrome expression at the gene level, two degenerated oligonucleotides against well conserved amino acid regions were designed after an optimal alignment of the known phytochrome sequences. These primers were tested against DNA isolated from Arabidopsis thaliana, and the polymerase-chain reaction (PCR) products were cloned and sequenced. The DNA fragment produced with this method proved to be identical with a phytochrome-A-gene fragment from A. thaliana, and hence this fragment was used in further experiments to prove whether amplified DNA from fern species contains phytochrome-like DNA. With this procedure we successfully detected and cloned gene fragments both from gametophytes of Anemia phyllitidis and Dryopteris filix-mas, cultured for 7 days under vegetative conditions. In addition, poly(A)+ RNA was prepared from 7-day-old gametophytes of A. phyllitidis, induced to differentiate antheridia under generative conditions. This poly(A)+ RNA was transcribed into complementary DNA and used together with both phytochrome specific primers in a PCR experiment. We thereby obtained another DNA fragment. These data strongly suggest that A. phyllitidis has at least two phytochrome genes, and that at least one of them is expressed in light-grown gametophytes.
光敏色素控制着蕨类植物配子体形成和分化过程中的几个发育阶段，包括孢子萌发、配子体形态发生或性细胞分化。为了获得有关光敏色素氨基酸序列及其在基因水平上表达调控的信息，在对已知光敏色素序列进行最佳比对后，针对保守的氨基酸区域设计了两条简并寡核苷酸。用这些引物对从拟南芥中分离的DNA进行检测，然后将聚合酶链反应（PCR）产物进行克隆和测序。用这种方法产生的DNA片段被证明与来自拟南芥的光敏色素A基因片段相同，因此该片段被用于进一步实验，以证明蕨类植物物种扩增出的DNA是否包含类光敏色素DNA。通过这个程序，我们成功地从在营养条件下培养7天的薄叶铁角蕨和鳞毛蕨的配子体中检测并克隆了基因片段。此外，从在生殖条件下诱导分化出雄器的7日龄薄叶铁角蕨配子体中制备了聚腺苷酸加尾RNA（poly(A)+ RNA）。这种poly(A)+ RNA被转录成互补DNA，并在PCR实验中与两种光敏色素特异性引物一起使用。由此我们获得了另一个DNA片段。这些数据有力地表明，薄叶铁角蕨至少有两个光敏色素基因，并且其中至少有一个在光照下生长的配子体中表达。
