THE GA5 LOCUS OF ARABIDOPSIS-THALIANA ENCODES A MULTIFUNCTIONAL GIBBERELLIN 20-OXIDASE - MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION
THE GA5 LOCUS OF ARABIDOPSIS-THALIANA ENCODES A MULTIFUNCTIONAL GIBBERELLIN 20-OXIDASE - MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION
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DOI：
10.1073/pnas.92.14.6640
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发表时间：
1995-07-03
影响因子：
11.1
通讯作者：
ZEEVAART, JAD
中科院分区：
文献类型：
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作者：
XU, YL;LI, L;ZEEVAART, JAD
GA(12)-醛后赤霉素（GAs）的生物合成涉及一系列氧化步骤，导致生物活性GAs的形成。先前，一个编码GA 20-氧化酶的cDNA克隆[赤霉素，2-氧葡萄糖酸酯：氧氧化还原酶（20-羟化，氧化），EC 1.14.11]。通过免疫筛选从南瓜（Cucurbita maxima L.）液体胚乳中提取的cDNA文库，获得了部分纯化的ga20 -氧化酶抗体。在这里，我们报道了从长日照物种拟南芥（Arabidopsis thaliana Heynh）的基因组文库中分离出GA 20氧化酶的基因组克隆。该基因组克隆包含一个由3个外显子和2个内含子组成的GA 20-氧化酶基因，这3个外显子长1131 bp，编码377个氨基酸残基，采用逆转录- pcr方法构建了与推定的GA 20-氧化酶基因组序列相对应的cDNA克隆。通过分析在大肠杆菌中表达的融合蛋白将GA（53）转化为GA（44）和GA（19）转化为GA（20）的能力，证实了cDNA克隆的身份。在衍生氨基酸水平上，拟南芥GA 20-氧化酶与南瓜GA 20-氧化酶的相似性为55%，相似性为80%。两种GA - 20氧化酶与其他2-氧戊二酸依赖的双加氧酶具有较高的同源性（2-ODDs），但两种GA - 20氧化酶的同源性最高。定位结果表明，克隆的ga20氧化酶与拟南芥GA5位点有紧密的连锁关系。ga5半矮突变体包含一个G -> a点突变，该突变在蛋白质编码序列中插入一个翻译终止密码子，从而证实ga5位点编码GA 20氧化酶。拟南芥叶片中GA5基因的表达在植株由短日照转为长日照后增强；它被GA(4)处理降低，表明终产物在GA生物合成途径中受到抑制。
The biosynthesis of gibberellins (GAs) after GA(12)-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe, This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns, The three exons are 1131-bp long and encode 377 amino acid residues, A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone,vas confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA(53) to GA(44) and GA(19) to GA(20). The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G --> A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA(4) treatment, suggesting end-product repression in the GA biosynthetic pathway.