Martin D N, Proebsting W M, Hedden P
Department of Horticulture and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331, USA.
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8907-11. doi: 10.1073/pnas.94.16.8907.
The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3beta-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3beta-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le "mutation" and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 microM and 13 microM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3beta-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3beta-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.
控制豌豆（Pisum sativum L.）茎伸长的主要赤霉素（GA）是GA1，它由GA20通过3β-羟基化形成。这一步骤限制了豌豆中GA1的生物合成，受Le位点控制，Le位点是最初的孟德尔位点之一。该位点的突变会导致植株矮化。我们从携带Le、le、le - 3和led等位基因的豌豆品系中分离出了编码GA 3β-羟化酶的cDNA。le和le - 3的cDNA序列各自包含一个碱基替换，相对于Le的序列导致单个氨基酸变化。led是从le品系衍生而来的突变体，其cDNA序列既包含le的“突变”，又有一个单碱基缺失，这导致阅读框移位，推测为无效突变。每个品系的cDNA在大肠杆菌中表达。来自Le的克隆的表达产物将GA9转化为GA4，将GA20转化为GA1，Km值分别为1.5 microM和13 microM。来自le的克隆中的氨基酸替换使GA9的Km增加了100倍，并将GA20的转化率降低到几乎为零。来自le和le - 3的表达产物具有相似水平的3β-羟化酶活性，而来自led的表达产物无活性。我们的结果表明3β-羟化酶cDNA由Le编码。Le转录本在发芽豌豆幼苗的根、茎和子叶、已定植幼苗的节间和叶片以及发育中的种子中表达。
