Oommen A, Dixon R A, Paiva N L
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402.
Plant Cell. 1994 Dec;6(12):1789-1803. doi: 10.1105/tpc.6.12.1789.
In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway.
在豆科植物中，感染和激发子诱导型抗菌植物抗毒素的合成是通过苯丙烷类途径的异黄酮分支进行的。为了研究异黄酮途径特异性基因的转录调控，我们分离了编码异黄酮还原酶（IFR）的基因，该酶催化苜蓿中植物抗毒素美迪紫檀素合成的倒数第二步反应。构建了IFR基因765bp和436bp启动子片段与β-葡萄糖醛酸酶报告基因的嵌合基因融合体，并通过农杆菌介导的转化将其导入苜蓿和烟草。两个启动子片段在来自这两个物种转基因植物的细胞悬浮培养物中均赋予激发子介导的表达，在转基因苜蓿叶片中赋予真菌感染介导的表达。在转基因苜蓿中，两个启动子片段指导的发育表达仅在根分生组织、皮层和根瘤中观察到，这与内源性IFR转录本的积累一致。然而，在转基因烟草中，765bp启动子的表达在营养组织（根分生组织和皮层、茎和叶柄的内部维管组织、叶尖以及叶柄附近的茎周边）和生殖组织（柱头、胎座、子房基部、花托、种子、绒毡层和花粉粒）中都能观察到，而436bp启动子仅在果实、种子和花粉中表达。这些数据表明，两个物种中IFR启动子的感染/激发子诱导性以及苜蓿中的发育表达是由-436位下游的序列决定的，而-436和-765之间的序列在缺乏异黄酮途径的异源物种中赋予了一种复杂的强异位发育表达模式。
