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

[807145]鲍曼，paul蚜虫是重要农业植物的主要害虫。基本上所有蚜虫都在细胞内的原核共生体（属于蚜虫属）中有特殊的细胞。内共生体不能在蚜虫体外生长。蚜虫依赖布氏虫；消灭内共生体会导致蚜虫死亡。蚜虫的食物植物汁液富含碳水化合物，但缺乏氨基酸。昆虫需要十种预先形成的氨基酸，人们认为内共生体的功能之一就是为蚜虫合成这些氨基酸。研究者过去的研究，以及其他研究者的研究，已经提供了内共生体为宿主制造必需氨基酸色氨酸的证据。从先前的研究中有营养证据表明，这种内共生体还能合成必需的支链氨基酸（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中常见的几种酶。亮氨酸途径起源于缬氨酸途径的中间体。其他研究人员发现，在一些蚜虫的布氏体中，亮氨酸生物合成的基因位于一个质粒上。本研究的目的是提供证据是否Buchnera参与支链氨基酸的生物合成。在本项目中，研究者将使用稻谷裂蚜（Schizaphis graminurn）和稻谷裂蚜（Diuraphis noxia）这两种谷物害虫。比较这两种蚜虫的Buchnera是有意义的，因为以前的研究表明，在Diuraphis noxia蚜虫中，Buchnera的生物合成活性需求较少。由于Buchnera不能在蚜虫外培养，研究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酶的基因是否存在于蚜虫S. graminum和D. noxia的Buchnera中。这将涉及克隆和测序内共生DNA片段杂交探针这些基因。随后，将对内生共生体进行纯化，并检测支链生物合成途径中选定酶的活性。使用定量逆转录酶聚合酶链反应，研究者的实验室还将确定编码这些选定酶的信使RNA （mRNA）的水平。预计关键调控酶的活性和mRNA水平会增加。此外，稻瘟病菌中布氏菌酶的比活性和mRNA水平可能高于稻瘟病菌中布氏菌酶的比活性。对内共生关联和氨基酸过量生产的一种可能的适应是关键调节酶对最终产物反馈抑制的脱敏。这些关键的布氏菌酶基因在大肠杆菌和纯化酶中会过表达；它们的动力学性质，相对于底物和最终产物抑制剂，将被确定。由于布氏菌对蚜虫的生存至关重要，有关其功能、遗传和生理的信息可能为控制蚜虫种群提供一种手段。研究蚜虫内生共生体对支链氨基酸的生物合成，可能会发现干扰这些必需氨基酸的生物合成的方法，从而减少蚜虫在重要农业植物上的数量。这可能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.