Plant Storage Lipid Biosynthesis

收入增加、人口增长以及对可再生液体燃料的迫切需求正在推动全球对种子油的前所未有的需求。据估计，今年加拿大将生产约1600万公吨油菜籽，为国家创造约190亿美元的经济活动（加拿大油菜籽理事会）。我的研究计划的总体目标是了解如何控制种子油的形成，作为开发具有增加的种子油含量和可被指定为“零饱和”的油的卡诺拉的基础，以提高油的竞争力和适销性。一般来说，饱和脂肪的消费增加往往与心血管疾病的患病率增加有关。在种子发育过程中，一系列的油脂生成酶（生物催化剂）驱动油的形成。这些酶的作用可以影响形成的油的量以及油的组成和性质。拟议的研究计划侧重于两种类型的酶，驱动石油建设途径中的反应。这些酶中的一种（缩写为DGAT）驱动油形成的最后步骤，而另一种酶（缩写为拉克）驱动用于最终油构建步骤的结构单元的形成。重组DNA技术将用于修饰卡诺拉DGAT，并将对所产生的酶的修饰形式进行生化测试。酶修饰将涉及从酶中去除部分或在酶内进行特定的改变。这种类型的探测应该提供一些关于酶如何运作的见解，并且能够容纳某些石油构建模块。高通量方法将用于产生多种酶变体，其中一些可能具有所寻求的特征。功能性DGAT变体的产生和鉴定将使用酵母系统进行，而在拉克的情况下，细菌系统将用于此目的。将选择驱动饱和度降低的油形成的DGAT变体进行进一步的生化测试。在拉克的情况下，将选择驱动具有降低的饱和度的石油结构单元的形成的酶变体。然后，通过基因工程将有希望的DGAT或拉克变体引入到模型油籽植物（塔勒水芹）中，以观察油中的饱和度是否降低。然后将评估降低塔勒水芹籽油中饱和度的酶变体降低芥花油中饱和度的能力。我们还将尝试纯化，结晶和确定卡诺拉DGAT的详细结构，以找出真正的“使它滴答作响”。如果我们工作的这个高风险部分成功，它将补充我们在DGAT修改方面的工作。目前，还没有关于这种酶的详细结构信息。关于这种关键的油脂生成酶的结构和作用的详细信息将导致开发用于修改种子油生产的其他策略。这种植物油形成的研究将依赖于许多学科的方法，包括植物生物化学，结构生物学，分子育种，分子生物学和遗传工程。拟议的研究计划也将有助于在植物油研究的新兴领域培养高素质的人才。

Rising incomes, population growth, and an urgent need for renewable liquid fuels are driving an unprecedented global demand for seed oils. This year it is estimated that Canada will produce about 16 million metric tonnes of canola generating about $19 billion in economic activity for the nation (Canola Council of Canada). The overall goal of my research program is to understand how seed oil formation is controlled as a basis for developing canola with increased seed oil content and oil that can be designated as “zero saturate” to increase both the competitiveness and marketability of the oil. In general, increased consumption of saturated fats tends to be associated with increased prevalence of cardiovascular disease. During seed development, a series of oil-building enzymes (biological catalysts) drive the formation of oil. The action of these enzymes can influence both the quantity of oil formed and the composition and properties of the oil. The proposed research program focuses on two types of enzymes that drive reactions in the oil-building pathway. One of these enzymes (abbreviated DGAT) drives the final step in oil formation whereas the other enzyme (abbreviated LACS) drives the formation of building blocks for use in the final oil-building step. Recombinant DNA technology will be used to modify canola DGAT and the resulting modified forms of the enzyme will undergo biochemical testing. Enzyme modification will involve removing sections from the enzyme or making specific changes within the enzyme. This type of probing should provide some insight into how the enzyme operates and is able to accommodate certain oil-building blocks. High throughput methods will be used to generate a multitude of enzyme variants, some of which may have the characteristics sought. Production and identification of functional DGAT variants will be conducted using a yeast system whereas a bacterial system will be used for this purpose in the case of LACS. DGAT variants which drive the formation of oils reduced in saturation will be selected for further biochemical testing. In the case of LACS, enzyme variants will be selected which drive the formation of oil-building blocks with reduced saturation. Promising DGAT or LACS variants will then be introduced into a model oilseed plant (thale cress), via genetic engineering, to see if the degree of saturation is reduced in the oil. Enzyme variants which reduce saturation in thale cress seed oil will then be evaluated for their ability to reduce saturation in canola oil. We will also attempt to purify, crystallize and determine the detailed structure of canola DGAT in order to find out what really “makes it tick”. If this high risk component of our work is successful, it will complement our work on DGAT modification. Currently, there is no detailed structural information available on this enzyme. Detailed information on the structure and action of this key oil-building enzyme will lead to the development of other strategies for modifying seed oil production. This plant oil formation research will rely on methods from numerous disciplines including plant biochemistry, structural biology, molecular breeding, molecular biology and genetic-engineering. The proposed research program will also contribute to the training of highly qualified personnel in the burgeoning area of plant oils research.