Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development
Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development
复制标题
DOI：
10.1023/a:1005715722193
复制
发表时间：
1997-04-01
影响因子：
5.1
通讯作者：
Hedden, P
中科院分区：
文献类型：
--
作者：
GarciaMartinez, JL;LopezDiaz, I;Hedden, P
根据赤霉素（GA）20-氧化酶的保守氨基酸序列设计简并引物，利用PCR技术从豌豆（Pisum sativum）幼子和菜豆（Phaseolus vulgaris）发育胚中分离出GA 20-氧化酶的cDNA克隆。从豌豆中获得了一个GA 20-氧化酶cDNA（Ps27-12），从豆类中获得了三个GA 20-氧化酶cDNA（Pv 15 -11、Pv 73 -1和Pv 85 -26）。通过证明在大肠杆菌中表达的融合蛋白表现出GA 20-氧化酶活性，将[C-14]GA（12）转化为[C-14]GA（9），证实了它们的身份。该三步反应中的中间体GA（15）和GA（24）也被鉴定为产物。三个克隆的表达蛋白（Pv 27 -12、Pv 15 -11和Pv 73 -1）也显示将GA（53）转化为GA（20），与它们转化GA（12）一样有效。基于通过北方印迹分析测量的转录水平，豌豆GA 20-氧化酶基因在幼叶、完全展开的节间、非常年轻的种子（直到开花后4天）和扩展的豆荚（从开花后3天至少到第6天）。在3日龄未授粉子房的豆荚中的表达高于授粉子房。用GA（3）处理未授粉子房以诱导单性结实坐果，严重降低了GA 20-氧化酶mRNA的量，而用2：4-D处理，虽然诱导坐果，但没有降低这些转录本的水平。在未授粉子房上方的植物去顶导致豆荚中非常高水平的GA 20-氧化酶mRNA。3个来自菜豆的GA 20-氧化酶基因表现出非常不同的表达模式：PV 15 -11在根、幼叶和发育中的种子中表达，但在未成熟的子叶中表达最高。而Pv 73 -1具有与Ps27-12相似的表达模式，其转录物仅在幼种子和幼叶中发现，在那里其特别丰富。在发育中的种子中检测到对应于Pv 85 -26的转录物，并且在幼叶中仅检测到痕量。Southern杂交分析表明，豆GA 20氧化酶是由单拷贝基因编码，而一个多基因，同源的Ps27-12，也可能存在于豌豆。
PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgar-is). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [C-14]GA(12) to [C-14]GA(9). The intermediates in this three-step reaction, GA(15) and GA(24), were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA(53) to GA(20), as effectively as they did GA(12) On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA(3) to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2:4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv15-11 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons. while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis indicated that the bean GA 20-oxidases are each encoded by single-copy genes, whereas one more gene, homologous to Ps27-12, could also exist in pea.