STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .11. MOLECULAR-CLONING AND EXPRESSION OF ALFALFA ISOFLAVONE REDUCTASE, A KEY ENZYME OF ISOFLAVONOID PHYTOALEXIN BIOSYNTHESIS
STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .11. MOLECULAR-CLONING AND EXPRESSION OF ALFALFA ISOFLAVONE REDUCTASE, A KEY ENZYME OF ISOFLAVONOID PHYTOALEXIN BIOSYNTHESIS
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DOI：
10.1007/bf00037051
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发表时间：
1991-10-01
影响因子：
5.1
通讯作者：
DIXON, RA
中科院分区：
文献类型：
--
作者：
PAIVA, NL;EDWARDS, R;DIXON, RA
苜蓿中的主要植物抗毒素是异黄酮类 (-)-Medicarpin（或 6aR、11aR）-Medicarpin。 异黄酮还原酶 (IFR) 是麦果品生物合成中的倒数第二个酶，负责在 (-)-麦果品中引入两个手性中心之一。 我们分离了 1.18 kb 的苜蓿 cDNA (pIFRalfl)，当在大肠杆菌中表达时，它可将 2'-羟基芒柄花素立体特异性转化为 (3R)-vestitone，正如对苜蓿 IFR 的预测一样。 源自 954 bp 开放阅读框的多肽的计算分子量 (35 400) 与从其他豆科植物纯化的 IFR 蛋白测定的估计 M(r) 相当。 转录本 (1.4 kb) 在诱导的苜蓿细胞培养物中被高度诱导。 诱导动力学与 IFR 活性的出现、Medicarpin 的积累以及观察到的途径中其他酶的诱导一致。 在健康的植物部分（根和根瘤）中发现了低水平的 IFR 转录本，这些部分积累了低水平的美果苷葡萄糖苷。 IFR 似乎是由苜蓿中的单个基因编码的。 IFR 的克隆开辟了通过改变异黄酮立体化学对苜蓿植物抗毒素生物合成进行遗传操作的可能性。
The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalfl) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35 400) derived from the 954 bp open reading frame compares favorably to estimate M(r)s determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.