Université François Rabelais de Tours, EA 2106 Biomolécules et Biotechnologies Végétales, IFR 135 Imagerie fonctionnelle, Tours, France.
Plant Biol (Stuttg). 2011 Jan;13(1):36-41. doi: 10.1111/j.1438-8677.2010.00325.x.
Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.
近年来，人们广泛研究了钙离子信号在长春花中单萜吲哚生物碱（MIA）生物合成调控中的作用，尽管尚未分离出该信号通路的蛋白质。本研究利用 PCR 策略，克隆并命名了两个编码不同钙调蛋白（CAM）同工型的长春花 cDNA，分别为 CAM1 和 CAM2。推导的 149 个氨基酸序列具有四个 Ca2+结合结构域，与拟南芥 CAM 同工型具有高度相似性（>91%）。表达相应的重组蛋白后，证明了 CAM1 和 CAM2 结合 Ca2+的能力。此外，CAM1-GFP 和 CAM2-GFP 在长春花细胞中的瞬时表达显示两种 CAM 均具有典型的核质定位，这与 CAM 靶蛋白的广泛分布一致。通过 RNA 印迹分析，我们表明 CAM1 和 CAM2 基因在长春花器官中有广泛的表达模式，并在长春花细胞培养周期中持续表达，而生长素对 CAM1 有轻微的抑制作用。通过 RNA 原位杂交，我们还在幼苗子叶的维管束区域检测到了 CAM1 和 CAM2 mRNA。最后，通过使用特异性抑制剂，我们还表明 CAM 通过作用于催化 MIA 生物合成早期步骤的酶编码基因的表达调控，在长春花细胞中需要 MIA 生物合成，例如 1-脱氧-d-木酮糖 5-磷酸还原异构酶和香叶醇 10-羟化酶。
