Plant Storage Lipid Biosynthesis

不断增长的收入、人口增长和对可再生液体燃料的迫切需求正在推动全球对种子油前所未有的需求。据估计，今年加拿大将生产约1600万公吨油菜籽，为国家带来约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，以此为基础开发种子油含量更高的油菜籽和可被指定为“零饱和”的油料，以提高油料的竞争力和适销性。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形成的油量，也可以影响油的组成和性质。拟议的研究计划重点放在两种类型的酶上，这两种酶推动了石油生成途径中的反应。其中一种酶(缩写为DGAT)驱动石油形成的最后一步，而另一种酶(缩写为LACS)驱动积木的形成，用于最终的制油步骤。重组DNA技术将被用于修饰油菜籽DGAT，由此产生的修饰形式的酶将接受生化测试。酶的修饰将包括从酶中移除部分或在酶中进行特定的改变。这种类型的探测应该能提供一些关于酶是如何工作的洞察力，并能够容纳某些石油构建块。高通量方法将被用来产生多种酶变体，其中一些可能具有所寻求的特征。功能性DGAT变异体的生产和鉴定将使用酵母系统进行，而对于LACS，将使用细菌系统进行这一目的。DGAT变种将被选择用于进一步的生化测试，这些变种导致油的饱和度降低。在LACS的情况下，将选择酶变体来驱动饱和度降低的石油构建块的形成。有希望的DGAT或LACS变种将通过基因工程被引入到模式油籽植物(水芹)中，以观察油中的饱和度是否降低。降低水芹籽油中饱和度的酶变种将被评估它们降低菜籽油中饱和度的能力。我们还将试图纯化、结晶和确定油菜籽DGAT的详细结构，以找出真正的“让它运转”的原因。如果我们工作中的这一高风险部分成功，它将补充我们在修改DGAT方面的工作。目前，还没有关于这种酶的详细结构信息。关于这种关键产油酶的结构和作用的详细信息将导致开发其他改良种子油生产的策略。这项植物油形成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、分子生物学和基因工程。拟议的研究计划还将有助于在新兴的植物油研究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.