Phillips A L, Huttly A K
Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, UK.
Plant Mol Biol. 1994 Feb;24(4):603-15. doi: 10.1007/BF00023557.
The Arabidopsis ga1 mutant has very low levels of endogenous, active gibberellins and thus has an extreme dwarf phenotype; application of GA3 induces stem elongation and flower development. To test the hypothesis that GA action in this system involves changes in gene expression, we have cloned mRNAs whose abundance changes following GA application. A subtraction cloning scheme for the isolation of differentially regulated cDNAs was established, involving hybridization of single-stranded cDNA to biotinylated mRNA. cDNA populations enriched up to 150-fold in GA-regulated sequences were produced and cDNA libraries generated. Screening of these libraries has isolated two clones that identify mRNAs of ca. 1100 and 750 bases whose abundance is markedly increased 24 h after GA application. One of these clones encodes the vegetative form of the Arabidopsis tonoplast intrinsic protein (gamma-TIP), a water channel protein, the expression of which has recently been shown to be correlated with regions of cell expansion. The second clone is expressed only in the inflorescence and encodes a proline- and glycine-rich protein that may be a cell wall component.
拟南芥ga1突变体的内源活性赤霉素水平极低，因此具有极端矮化的表型；施用GA3可诱导茎伸长和花发育。为了验证该系统中GA作用涉及基因表达变化这一假说，我们克隆了施用GA后丰度发生变化的mRNA。建立了一种用于分离差异调节cDNA的消减克隆方案，包括将单链cDNA与生物素化mRNA杂交。产生了在GA调节序列中富集高达150倍的cDNA群体，并构建了cDNA文库。对这些文库的筛选分离出了两个克隆，它们鉴定出约1100和750个碱基的mRNA，其丰度在施用GA后24小时显著增加。其中一个克隆编码拟南芥液泡膜内在蛋白（γ-TIP）的营养型，γ-TIP是一种水通道蛋白，最近已证明其表达与细胞扩张区域相关。第二个克隆仅在花序中表达，编码一种富含脯氨酸和甘氨酸的蛋白，可能是一种细胞壁成分。
