Molecular Dissection of Metabolic Channels for Sterol and Sesquiterpene Metabolism in Tobacco

异戊二烯类化合物在自然界中普遍存在，是生命所必需的，并调节着生物体与其环境之间的许多相互作用。此外，植物是药用上重要的异戊二烯的丰富来源。然而，我们对植物中类异戊二烯生物合成途径的了解是有限的。最近的研究表明，烟草细胞悬浮培养特别适合于对异戊二烯代谢中一个重要但可能的分支点进行详细的生理和生化研究。当真菌诱导子被添加到细胞培养物中时，培养物停止生产甾醇，而是合成和分泌抗微生物倍半萜类化合物。甾醇生物合成的下降与角鲨烯合成酶活性的抑制和倍半萜环化酶诱导倍半萜类生物合成有关。由于这两种酶位于该途径中假定的分支点，因此一种酶的诱导和另一种酶的抑制被解释为控制碳流从而最终产物形成的重要调控机制。这一解释表明，类异戊二烯代谢发生在一个均匀的环境中，中间体自由混合，其他连续或竞争的酶可以接触到。最近的结果表明并非如此，并表明专门用于生产特定最终产品的独立调节的同工酶阵列可能沿着表面或膜排列。这一离散代谢通道假说预测，在激发子处理的细胞中，编码倍半萜生物合成酶的基因与对照细胞中的固醇代谢基因相比，具有选择性转录，并且酶蛋白必须具有靶向信息才能插入正确的代谢通道或单位。这项工作的目标是使用分子方法来验证这些预测，包括倍半萜环酶和角鲨烯合成酶基因的克隆，对指导它们表达的顺式元件的分析，以及将它们可能的靶向信号融合到一个报告基因上，这将改变形成的异戊二烯的种类或数量。这些研究的结果将有助于阐明植物体内异戊二烯代谢的调控机制，并将对未来利用基因工程技术操纵这一途径产生重要影响。

Isoprenoids are ubiquitous in nature, essential for life, and mediate a number of interactions between organisms and their environment. In addition, plants are a rich source for pharmaceutically important isoprenoids. Yet, our understanding of the isoprenoid biosynthetic pathway in plants is limited. Recent studies indicate that tobacco cell suspension cultures are uniquely suited for detailed physiological and biochemical studies of one important but putative branch point in isoprenoid metabolism. When fungal elicitors are added to the cell cultures, the cultures cease sterol production and instead synthesize and secrete anti-microbial sesquiterpenoids. The decline in sterol biosynthesis has been correlated with suppression of squalene synthetase enzyme activity, and the induction of sesquiterpenoid biosynthesis wit the induction of a sesquiterpene cyclase. Because these two enzymes are positioned at a putative branch point in the pathway, the induction of one enzyme and the suppression of the other were interpreted as an important regulatory mechanism controlling carbon flow and hence, end product formation. This interpretation suggested that isoprenoid metabolism was occurring in a homogeneous environment with intermediates mixing freely and accessible to other to successive or competing enzymes. Recent results suggest otherwise and indicate that independently regulated arrays of isozymes dedicated to the production of specific end products might be aligned along a surface or membrane.. This hypothesis of discrete metabolic channels predicts selective transcription of genes coding for enzymes of sesquiterpene biosynthesis in elicitor-treated cells versus genes for sterol metabolism in control cells, and that the enzyme proteins must have targeting information for insertion into the correct metabolic channel or unit. The goals of this work are to test these predictions using a molecular approach including cloning of the sesquiterpene cyclase and squalene synthetase genes, analysis of the cis-elements directing their expression, and fusion of their putative targeting signal onto a reporter gene which will either alter the kind or amount of isoprenoid formed. Results from these studies will help elucidate the mechanisms regulating isoprenoid metabolism in plants, and will have important impact on for future manipulations of this pathway using genetic engineering technology.//