Study of an Enzyme that Regulates Oil Synthesis in Seeds

三酰甘油（TAG）在种子成熟和萌发过程中的合成和降解涉及活跃的代谢途径。了解TAG代谢的详细机制将有助于控制植物生命周期活动阶段的食物储备沉积和利用。它还将为如何培育或基因工程更好地为人类利用种子油提供不可或缺的信息。实验室的长期目标是阐明种子中TAG代谢的机制。重点是一个关键的TAG合成酶，溶血磷酸酯（LPA）酰基转移酶，在成熟的种子。这种酶催化酰基辅酶a的酰基部分结合到1-酰基甘油-3-磷酸的sn-2位置，产物磷脂酸将进一步代谢为TAG或磷脂。该酶对这两种底物都有非常严格的酰基偏好，因此控制了首选脂肪酸进入TAG的sn-1和sn-2位置。脂肪酸组成以及种子TAG的性质在很大程度上依赖于LPA酰基转移酶的酰基偏好。在这个研究计划中，将追求以下两个目标。第一个目的是研究一种植物的特殊酰基偏好的种子LPA酰基转移酶在遗传转化后是否会在另一种植物中活跃。实验将对棕榈、芸苔属植物和绵草属植物的种子LPA酰基转移酶进行表征，获得它们转化相应植物的基因，并检测转化植物中外源酶的活性。将使用几种不同的方法来获得该基因；这些方法包括：通过从纯化酶和细菌基因中推断出的寡核苷酸筛选基因cDNA文库，并与细菌突变体进行功能互补。已经获得了与编码该酶的植物cDNA功能互补的推定大肠杆菌突变系；这几行将得到最深入的研究。如果时间允许，我们将利用棕榈和meadowfoam的LPA酰基转移酶基因对芸苔进行转化，并分析转化植株的外源酶活性。这些信息将对我们理解脂肪酸在种子中组装成TAG的控制机制以及非种子器官中的甘油脂的控制机制至关重要。第二个目的是测试细菌来源的膜相关脂质代谢酶是否在高等植物中具有活性。利用编码LPA酰基转移酶的大肠杆菌基因转化芸苔，并研究该细菌基因在转化芸苔中的表达。这个项目的目的是提取一种基因，这种基因可以产生一种酶，这种酶可以产生棕榈树中发现的某种植物油，并将这种基因引入其他植物中，看看它们是否也能生产棕榈油。如果这是成功的，研究人员已经找到了一种方法，使在美国不常见的植物中产生的油可以在美国其他常见的植物中得到。此外，这些酶还可用于制备新型脂质生物材料。

Synthesis and degradation of triacylglycerols (TAG) in seeds during maturation and germination involve active metabolic pathways. Understanding the detailed mechanism of TAG metabolism will enable the control of food reserve deposition and utilization during active stages of the plant life cycle. It will also provide an indispensable information of how to breed or genetically engineer better seed oils for human utilization. The long term objective of the laboratory is to elucidate the mechanism of TAG metabolism in seeds. The focus is on a crucial TAG synthesizing enzyme, lysophosphatidate (LPA) acyltransferase, in maturing seed. This enzyme catalyzes the incorporation of acyl moiety from acyl CoA to the sn-2 position of 1-acyl-glycerol-3-phosphate, and the product phosphatidate will be further metabolized to either TAG or phospholipids. The enzyme has a very stringent acyl preference on both substrates, and thus controls the incorporation of preferred fatty acids into the sn-1 and sn-2 positions of TAG. The fatty acid composition, and thus the properties of seed TAG, rely heavily on the acyl preference of LPA acyltransferase. In this research proposal, the following two objectives will be pursued. The first objective is to examine whether seed LPA acyltransferase of a special acyl preference from one plant species will be active in another plant species after genetic transformation. Experiments will be performed to characterized seed LPA acyltransferases from palm, Brassica, and meadowfoam, to obtain their genes for transformation of appropriate plants, and to detect activities of the foreign enzymes in the transformed plants. Several different approaches will be used to obtain the gene; these approaches include: screening cDNA libraries for the genes by oligonucleotides deduced from the purified enzyme and by a bacterial gene, and functional complementation with a bacterial mutant. Putative E. coli mutant lines functionally complemented with plant cDNA encoding the enzyme have been obtained; these lines will be studied most intensively. If time permits, we will use the palm and meadowfoam LPA acyltransferase genes to transform Brassica, and analyze the transformed plants for activities of the foreign enzyme. The information will be crucial to our understanding of the control mechanism of fatty acid assembly into TAG in seeds as well as glycerolipids in non seed organs. The second objective is to test if a membrane-associated, lipid-metabolizing enzyme of bacterial origin can be active, in higher plants. by transforming Brassica with an E. coli gene encoding LPA acyltransferase and studying the expression of the bacterial gene in the transformed Brassica. %%% This project aims to take a gene which makes an enzyme that Produces a certain plant oil found in palms, and introduce the gene into other plants to see if they also would produce the palm oil. If this is successful the investigators have found a way for oil produced in plants not common to the US to be available in other species of plants common in this country. Furthermore, these enzymes could be used to make novel lipid biomaterials.