Enzymatic and molecular bases of trade-offs in lipid metabolism that underlie a life history trade-off.

这个项目的最终目标是确定在中间代谢中的基于基因的修饰，这些修饰是生活史特征中基于基因的变异的基础。实验将在有翼多态蟋蟀（Gryllus firmus）中进行，这种蟋蟀有飞行能力，但会推迟产卵时间，而没有飞行能力，但产卵量会大大增加。先前的研究表明，在具有飞行能力的形态中，产卵量减少是因为大量的甘油三酯飞行燃料必须积累。先前的研究也证明，甘油三酯积累的增加是由于脂肪酸生物合成的增加和脂肪酸转向生产甘油三酯而不是磷脂的增加。拟议的研究将建立在这个广泛的数据库上，并有两个目标：第一是确定具有飞行能力的形态中负责增加脂肪酸生物合成的酶学特征和基因调控机制。第二个目标是确定甘油三酯/磷脂生物合成分支点酶的功能修饰，导致脂肪酸优先转移（即权衡）为甘油三酯飞行燃料。四种关键的脂质生物合成酶将被纯化至均匀性，针对它们产生抗体，然后用于测量酶的浓度。这将验证脂质生物合成酶的酶活性升高是由于酶浓度升高的假设。纯化酶将被动力学表征，以测试另一种假设，即酶活性的形态差异是由催化效率的变化引起的，这是由于不同磷酸化等因素造成的。将比较编码四种脂肪生成酶的基因的信息丰度，以确定酶活性/浓度的形态差异在多大程度上是由基因表达差异引起的。最后，通过甘油三酯/磷脂生物合成分支点控制通量的五种酶将在具有飞行能力和不具有飞行能力的变体之间进行比较，比较其各种功能特征，如比活性、差异磷酸化和与膜的结合。目标将是确定在能够飞行和不能飞行的形态中，负责脂肪酸转化为甘油三酯（飞行燃料）和磷脂（关键鸡蛋成分）的形态特异性权衡的酶的差异。这项研究将有助于我们理解生命史进化的功能原因，这是进化生物学中一个鲜为人知的话题。它将包括对中间代谢变化的最详细分析，这些变化引起了自然种群中发生的生活史变化和权衡。这将是第一个在中间代谢的分支点（甘油三酯/磷脂）确定特定酶改变的研究，这是生活史权衡的基础。这个项目将有助于生命史进化和进化生物化学的深度整合，这是在之前的nsf资助的研究中开始的。该项目还将通过为进化生物学、酶学和分子生物学的博士后提供综合培训，为人力资源的发展做出贡献，并将提供许多重要的本科生研究机会。

Enzymatic and molecular bases of trade-offs in lipid metabolism that underlie a life-history trade-off Anthony J. ZeraUniversity of Nebraska The ultimate goal of this project is to identify genetically-based modifications in intermediary metabolism that underlie genetically-based variation in life history traits. Experiments will be undertaken in the wing-polymorphic cricket, Gryllus firmus, which exists as a flight-capable morph that delays egg production and a flightless morph with substantially enhanced egg production. Previous studies demonstrated that egg production in the flight-capable morph is reduced because a large amount of triglyceride flight fuel must accumulate. Previous studies also have documented that the increased accumulation of triglyceride results from increased fatty-acid biosynthesis and increased diversion of fatty acids into the production of triglyceride as opposed to phospholipid. Proposed research will build on this extensive data base and has two goals: The first is to identify the enzymological characteristics and gene-regulatory mechanisms in the flight-capable morph responsible for the increased biosynthesis of fatty acid. The second goal is to identify functional modifications of enzymes at the triglyceride/ phospholipid biosynthetic branch point that result in the preferential diversion (i.e. trade-off) of fatty acid into triglyceride flight fuel in the flight-capable morph. Four key lipid-biosynthetic enzymes will be purified to homogeneity, antibodies will be raised against them, and then used to measure enzyme concentration. This will test the hypothesis that elevated enzyme activity of lipid-biosynthetic enzymes is due to elevated enzyme concentration. Purified enzymes will be characterized kinetically to test the alternate hypothesis that morph-differences in enzyme activities result from changes in catalytic efficiency, due to factors such as differential phosphorylation. Message abundance of genes encoding the four lipogenic enzymes will be compared to determine the extent to which morph-differences in enzyme activity/concentration result from differences in gene expression. Finally, five enzymes controlling flux through the triglyceride/phospholipid biosynthetic branch point will be compared between flight-capable and flightless morphs with respect to a variety of functional characteristics such as specific activity, differential phosphorylation, and binding to membranes. The goal will be to identify differences in the enzymes that are responsible for the morph-specific trade-off in the diversion of fatty acid into triglyceride (flight fuel) vs. phospholipid (key egg component) in flight-capable vs. flightless morphs.This study will contribute significantly to our understanding of the functional causes of life history evolution, a poorly understood topic in evolutionary biology. It will comprise the most detailed analysis of alterations in intermediary metabolism that give rise to life history variation and trade-offs that occur in natural populations. This will be the first study to identify specific enzymatic alterations at a branch point (triglyceride/phospholipid) in intermediary metabolism that underlies a life history trade-off. This project will contribute to the development of a deep integration of life history evolution and evolutionary biochemistry, which was initiated in the previous NSF-funded study. This project will also contribute to the development of human resources by providing integrative training for a postdoctoral associate in evolutionary biology, enzymology, and molecular biology, and will also provide numerous significant undergraduate research opportunities.