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空前需求。据估计，今年加拿大将生产约1600万吨油菜籽，为国家创造约190亿美元的经济活动（加拿大油菜籽委员会）。我的研究计划的总体目标是了解如何控制种子油的形成，作为开发增加种子油含量和可指定为“零饱和”油的菜籽油的基础，以提高油的竞争力和适销性。一般来说，饱和脂肪摄入量的增加往往与心血管疾病患病率的增加有关。在种子发育过程中，一系列造油酶（生物催化剂）驱动油的形成。这些酶的作用既可以影响油的形成量，也可以影响油的组成和性质。拟议的研究计划侧重于两种驱动油生成途径反应的酶。其中一种酶（缩写DGAT）驱动成油的最后一步，而另一种酶（缩写LACS）驱动最终成油步骤中使用的构建块的形成。重组DNA技术将用于修饰油菜籽DGAT，所得到的修饰形式将进行生化测试。酶修饰包括从酶上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洞察酶是如何运作的，以及能够容纳某些石油构建块。高通量方法将用于产生大量的酶变体，其中一些可能具有所寻求的特征。生产和鉴定功能性DGAT变体将使用酵母系统进行，而在LACS的情况下，将使用细菌系统进行此目的。驱动饱和度降低的油形成的DGAT变体将被选择进行进一步的生化测试。在LACS的情况下，酶变体将被选择，驱动形成油结构块与降低饱和度。然后，有希望的DGAT或LACS变体将通过基因工程引入模型油籽植物（泰利芥），以观察油中的饱和程度是否降低。然后将评估降低芥蓝籽油饱和度的酶变体降低菜籽油饱和度的能力。我们还将尝试纯化、结晶和确定油菜籽DGAT的详细结构，以找出真正“使其发挥作用”的原因。如果我们的工作中这个高风险的部分是成功的，它将补充我们对DGAT修改的工作。目前，还没有关于这种酶的详细结构信息。关于这种关键造油酶的结构和作用的详细信息将导致开发其他改变种子油产量的策略。这种植物油形成的研究将依赖于包括植物生物化学、结构生物学、分子育种、分子生物学和基因工程在内的众多学科的方法。该研究计划也将有助于培养新兴的植物油研究领域的高素质人才。

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.