Genetic Control of TCA Cycle During Seed Germination

成熟的油料种子（黄瓜、南瓜、花生、芸苔等）含有 线粒体少，三羧酸（TCA）水平低 循环酶 在吸胀之后，TCA循环酶被 从头合成。 TCA循环酶的生物发生， 然而，并不统一。 非脱羧部分 途径（琥珀酸和草酰乙酸之间的反应）是 首先合成。 几天后， 循环中的脱羧部分，丙酮酸 (or柠檬酸盐）和琥珀酸盐。 这一转变源于 改变这些种子子叶的新陈代谢。 年初 萌发时，子叶将储存的脂质转化为 糖类。 而TCA的非脱羧部分 循环对于该过程是必要的，脱羧部分是 不存在，从而防止通过脱羧的碳损失 反应. 后来，当这些组织开始进行光合作用时， 需要TCA循环，脱羧部分的酶 是合成的。 在这份提案中，奥利弗博士将研究 这种差异表达的机制， 蛋白质的合成是使用胰蛋白酶来完成的，胰蛋白酶是一种特异于 非脱羧部分和NAD+依赖性异柠檬酸 脱氢酶，一种仅在脱羧部分发现的酶 TCA循环。 蛋白质已经被纯化，抗体工具 正在产生，N-末端序列信息正在产生， 得到了 将使用抗体和/或寡核苷酸探针 为了分离这两个基因的cDNA（然后是基因组）克隆， proteins. 基因表达的控制水平（转录， 翻译等）将通过测量mRNA水平来确定， 进行转录检测。 启动子-GUS构建将是 转化到甘蓝型油菜中，这些转基因植物将 用于学习，1。时间进程和组织特异性 两个基因的表达，2。环境和生理 控制这两种基因表达的因子，以及3. 在种子成熟过程中抑制TCA循环蛋白。 启动子 分析将开始。 这些研究可能会得到加强， 使用瞬时表达测定和快速循环芸苔属或 拟南芥。

Mature oil seeds (cucumber, pumpkin, peanut, Brassica, etc) contain few mitochondria and have low levels of tricarboxylic acid (TCA) cycle enzymes. Following imbibition the TCA cycle enzymes are synthesized de novo. The biogenesis of the TCA cycle enzymes, however, is not uniform. The non-decarboxylative portion of the pathway (the reactions between succinate and oxaloacetate) is synthesized first. A few days later, the enzymes of the decarboxylative part of the cycle, the reactions between pyruvate (or citrate) and succinate, are formed. This switch results from changing metabolism in the cotyledons of these seeds. Early in germination the cotyledons are converting stored lipid to carbohydrate. While the non-decarboxylative portion of the TCA cycle is essential for this process the decarboxylative portion is not present thus preventing carbon loss through the decarboxylation reactions. Later as these tissues become photosynthetic, a full TCA cycle is needed and the enzymes of the decarboxylative portion are synthesized. In this proposal Dr. Oliver will examine the mechanism by which this differential expression of mitochondrial proteins is accomplished using fumarase, an enzyme specific for the non-decarboxylative part, and the NAD+-dependent isocitrate dehydrogenase, an enzyme found only in the decarboxylative portion of the TCA cycle. The proteins have been purified, antibody tools are being produced, and N-terminal sequence information is being obtained. The antibody and/or oligonucleotide probes will be used to isolate the cDNA (and then genomic) clones for these two proteins. The level of control of gene expression(transcriptional, translational, etc) will be determined by measuring mRNA levels and with run on transcription assays. Promoter-GUS contructs will be transformed into Brassica napus and these transgenic plants will be used to study, 1. the time course and tissue specificity of expression of both genes, 2. environmental and physiological factors that control the expression of these two genes, and 3. repression of TCA cycle proteins during seed maturation. Promoter analysis will be initiated. These studies may be augmented by the use of transient expression assays and rapid cycling Brassica or Arabidopsis.