Stiekema W J, Wimpee C F, Silverthorne J, Tobin E M
Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024.
Plant Physiol. 1983 Jul;72(3):717-24. doi: 10.1104/pp.72.3.717.
Hybridization probes for two nuclear-coded chloroplast proteins of Lemna gibba L. G-3 have been constructed in order to investigate phytochrome regulation of specific sequences. The first probe is a cDNA clone encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. This probe was isolated from a set of Lemna cDNA clones in the bacterial plasmid pBR322. The second probe is a subclone of a genomic clone encoding the light-harvesting chlorophyll a/b-protein. This clone was isolated from a set of genomic clones constructed in the lambda vector Charon 4 with L. gibba DNA fragments generated by partial EcoR1 digestion. The identity of these clones was confirmed by in vitro translation of RNA which hybridized to the cloned DNA. Plants grown under continuous white light contain high concentrations of both RNA sequences; however, when these plants are put into darkness the concentration of these RNAs decreases rapidly relative to the total amount of RNA. Plants grown in the dark with intermittent red light (2 minutes/8 hours) and put into complete darkness for 8 days also contain lower concentrations of the sequences in the total RNA. One minute of red light after this dark period results in a rapid increase in the levels of RNA hybridizing to the probes. The effect of red light can be reversed by far-red light. These experiments demonstrate that phytochrome action can rapidly influence either the rates of transcription or the rates of degradation of these mRNAs.
为了研究光敏色素对特定序列的调控，构建了浮萍（Lemna gibba L. G-3）两种核编码叶绿体蛋白的杂交探针。第一个探针是编码1,5-二磷酸核酮糖羧化酶小亚基的cDNA克隆。该探针是从细菌质粒pBR322中的一组浮萍cDNA克隆中分离得到的。第二个探针是编码捕光叶绿素a/b蛋白的基因组克隆的亚克隆。该克隆是从用部分EcoR1消化产生的浮萍DNA片段构建于λ载体Charon 4中的一组基因组克隆中分离得到的。通过与克隆DNA杂交的RNA的体外翻译证实了这些克隆的身份。在连续白光下生长的植物中这两种RNA序列的浓度都很高；然而，当这些植物置于黑暗中时，相对于RNA总量，这些RNA的浓度迅速降低。在黑暗中用间歇红光（2分钟/8小时）处理并在完全黑暗中放置8天的植物，其总RNA中这些序列的浓度也较低。在这段黑暗期后照射1分钟红光会导致与探针杂交的RNA水平迅速增加。红光的作用可以被远红光逆转。这些实验表明，光敏色素的作用可以迅速影响这些mRNA的转录速率或降解速率。
