Symbiotic Association of Aphids: Synthesis of Essential Amino Acids by the Nonculturable Prokaryotic Endosymbiont

9807145鲍曼、保拉麦是重要农业作物的主要害虫。基本上，所有的蚜虫在特化的细胞内都有原核共生体(归于Buchnera属)。内共生菌不能在蚜虫外生长。蚜虫依赖于Buchnera；内共生菌的消除会导致蚜虫的死亡。植物汁液是蚜虫的食物，含有丰富的碳水化合物，但缺乏氨基酸。昆虫需要10种预先形成的氨基酸，据认为，内共生体的功能之一是为蚜虫合成这些氨基酸。这位研究人员过去的研究以及其他研究人员的研究都提供了证据，证明内共生菌为宿主制造必要的氨基酸色氨酸。从以前的研究中有营养证据表明，内共生菌还合成必要的支链氨基酸(异亮氨酸、缬氨酸、亮氨酸)。支链氨基酸的生物合成途径涉及异亮氨酸和缬氨酸生物合成所共有的几种酶。亮氨酸途径起源于缬氨酸途径的中间产物。其他研究人员发现，在一些蚜虫的Buchnera中，亮氨酸生物合成的基因位于一个质粒上。这项研究的目的是为Buchnera是否参与支链氨基酸的生物合成提供证据。在这个项目中，研究人员将使用麦长管蚜和麦长管蚜，这两种害虫都是谷物的害虫。比较这两种蚜虫的Buchnera是有意义的，因为以前的研究表明，在Diuraphim noxia中对Buchnera的生物合成活性的需求较少。由于Buchnera不能在蚜虫以外的地方种植，调查者将使用几种方法来检查Buchnera是否为蚜虫宿主合成支链氨基酸。首先，实验室将确定编码支链氨基酸生物合成途径的酶的基因是否存在于棉铃虫中，这些基因来自麦长管蚜和新麦长管蚜。这将涉及克隆和测序内共生体DNA片段，这些片段与这些基因的探针杂交。随后，将对内生菌进行纯化，并检测支链生物合成途径中选定的酶的活性。利用定量逆转录聚合酶链式反应，研究人员的实验室还将确定编码这些选定酶的信使RNA(MRNA)的水平。关键调控酶的活性和信使核糖核酸水平有望提高。此外，禾谷链霉菌Buchnera酶的比活力和mRNA水平可能高于诺氏链霉菌Buchnera的酶活性和mRNA水平。一种可能的适应内共生结合和氨基酸过量生产的方法是使受调控的关键酶对最终产物反馈抑制脱敏。这些关键的Buchnera酶的基因将在大肠杆菌和纯化的酶中过表达；它们相对于底物和最终产物抑制剂的动力学特性将被确定。由于Buchnera对蚜虫的生存是必不可少的，关于内共生菌的功能、遗传学和生理学的信息可能提供控制蚜虫种群的手段。对蚜虫内共生体合成支链氨基酸的研究可能揭示出干扰这些必需氨基酸的生物合成的方法，从而减少农业上重要植物上的蚜虫种群。这可能会产生重大的经济影响，因为蚜虫和蚜虫传播的植物病毒使植物衰弱，大大降低了农业生产力。

9807145Baumann, PaulAphids are major pests of agriculturally important plants. Essentially all aphids harbor intracellular prokaryotic symbionts (assigned to the genus Buchnera) within specialized cells. The endosymbionts cannot be grown outside the aphid. Aphids are dependent on Buchnera; elimination of the endosymbionts results in death of the aphid. Plant-sap, the diet of aphids, is rich in carbohydrates but deficient in amino acids. Insects require ten preformed amino acids and it is thought that one of the functions of the endosymbiont is the synthesis of these amino acids for the aphid. The investigator's past studies, as well as those of other investigators, have provided evidence that the endosymbionts make the essential amino acid tryptophan for the host. From previous studies there is nutritional evidence that the endosymbiont also synthesizes the essential branched chain amino acids (isoleucine, valine, leucine). The pathway of branched chain amino acid biosynthesis involves several enzymes common to both isoleucine and valine biosynthesis. The leucine pathway originates from intermediates of the valine pathway. Other investigators have found that in the Buchnera of some aphids, the genes for leucine biosynthesis are located on a plasmid. The goal of this research is to provide evidence on whether Buchnera is involved in the biosynthesis of branched chain amino acids. In this project the investigator will use the aphids Schizaphis graminurn and Diuraphis noxia, both of which are pests of cereals. The comparisons of Buchnera from these two aphids is of interest since previous studies suggested that there is less demand for the biosynthetic activities of Buchnera in the aphid Diuraphis noxia. Since Buchnera cannot be cultivated outside the aphid, the investigator will use several approaches to examine whether Buchnera synthesizes branched-chain amino acids for the aphid host. Initially the laboratory will establish if the genes encoding enzymes of the branched chain amino acid biosynthetic pathway are present in Buchnera from the aphids S. graminum and D. noxia. This will involve cloning and sequencing endosymbiont DNA fragments which hybridize with probes for these genes. Subsequently, the endosymbionts will be purified and assayed for the presence of activities of selected enzymes of the branched chain biosynthetic pathway. Using the quantitative reverse transcriptase polymerase chain reaction, the investigator's laboratory will also determine the levels of messenger RNA (mRNA) encoding these selected enzymes. Increases in the activity and mRNA levels of key regulated enzymes are expected. In addition, the specific activities and the levels of mRNA for the enzymes from Buchnera from S. graminurn may be higher than those for the enzymes from Buchnera from D. noxia. One possible adaptation to an endosymbiotic association and overproduction of amino acids is desensitization of key regulated enzymes to end-product feedback inhibition. The genes for such key Buchnera enzymes will be overexpressed in Escherichia coli and enzymes purified; their kinetic properties, with respect to substrates and end-product inhibitors, will be determined.Since Buchnera is essential for the survival of aphids, information on the endosymbiont's function, genetics, and physiology may provide a means of controlling aphid populations. Research on the biosynthesis of branched chain amino acids by aphid endosymbionts may reveal ways in which it will be possible to interfere with the biosynthesis of these essential amino acids, thereby reducing aphid populations on agriculturally important plants. This could have a major economic impact since the debilitation of plants by aphids and by the plant viruses the aphids transmit significantly reduces agricultural productivity.