Lester D R, Ross J J, Davies P J, Reid J B
Department of Plant Science, University of Tasmania, Hobart, Australia.
Plant Cell. 1997 Aug;9(8):1435-43. doi: 10.1105/tpc.9.8.1435.
We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel. Heterologous screening of a pea cDNA library yielded a partial clone that was 61% identical to coding regions of the putative Arabidopsis gibberellin 3 beta-hydroxylase gene, GA4. DNA gel blot analysis with this cDNA revealed a HindIII restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus. Genomic clones of the GA4-related gene were isolated from the Le and le isolines. Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus. A cDNA containing a complete open reading frame of the pea GA4-related gene was amplified by polymerase chain reaction from each isoline. Recombinant expression in Escherichia coli demonstrated that the product of the Le cDNA was a gibberellin 3 beta-hydroxylase that is able to convert GA20 to the bioactive GA1. Substantially reduced levels of gibberellin 3 beta-hydroxylase activity were measured, after expression of the le cDNA, by using identical methods. This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.
我们描述了豌豆Le基因的分离过程,该基因控制节间伸长,最初由孟德尔描述。对豌豆cDNA文库进行异源筛选得到了一个部分克隆,它与拟南芥假定的赤霉素3β-羟化酶基因GA4的编码区有61%的同源性。用该cDNA进行DNA凝胶印迹分析,发现在孟德尔Le位点存在差异的豌豆近等基因系之间存在HindIII限制性片段长度多态性。从Le和le近等基因系中分离出了GA4相关基因的基因组克隆。利用聚合酶链反应结合限制性片段长度多态性分析表明该基因定位于Le位点。通过聚合酶链反应从每个近等基因系中扩增出了包含豌豆GA4相关基因完整开放阅读框的cDNA。在大肠杆菌中的重组表达表明,Le cDNA的产物是一种赤霉素3β-羟化酶,能够将GA20转化为生物活性GA1。在表达le cDNA后,用相同方法测定的赤霉素3β-羟化酶活性大幅降低。这种活性降低与该酶预测氨基酸序列中靠近其假定活性位点处的丙氨酸到苏氨酸的替换有关。