Symbiotic Association of Aphids: Biochemical Role of the Prokaryotic Endosymbiont

9402813鲍曼蚜虫的生存依赖于与蚜虫体腔内特殊细胞内的原核内共生体的关联。利用绿芽蚜虫（Schizaphis graminum），我们发现其内共生体（Buchnera aphidicola）具有色氨酸生物合成途径的所有基因，并且在质粒上扩增了trpEG（该途径的第一个酶——蒽醌合成酶的基因）。这些结果与过去的观察结果一致，表明内共生体合成色氨酸被蚜虫宿主利用。初步结果表明，在中国蚜虫（Schlectendalia chinensis）的远亲内共生体中，tryEG有一个染色体位置。我们打算通过检测该蚜虫种内共生体中色氨酸生物合成途径的基因组织，并调查确定trpEG染色体定位基因扩增在其他蚜虫种内共生体中的分布来继续这项研究。第一个反应由3-脱氧-d -阿拉伯糖-七磷酸（DAHP）合成酶催化，引发芳香氨基酸（色氨酸、苯丙氨酸、酪氨酸）和叶酸的生物合成。我们打算确定这个基因是否位于质粒上，在染色体上，或者是否有同功能的DAHP合成酶。这将通过限制性内切酶和Southern blot分析以及该酶基因的克隆和测序来完成。将与大肠杆菌和其他生物体中的基因组织进行比较。在大多数原核生物中，核糖体RNA的基因排列为一个单转录单位，顺序为16S和5S rnas。我们已经证明，蚜虫只有一个16S - 23s rRNA基因拷贝，并且不寻常的是它与其他rRNA基因不相连。我们将继续研究刺蚜23S和5S rRNA基因的组织结构。这些基因将被克隆、测序，并将上游和下游区域与假定的蚜虫16S rRNA转录单元的启动子和终止子进行比较。在它们生长最活跃的阶段，蚜虫通过孤雌生殖繁殖，产生活的幼虫。在蚜虫的生长周期中，关于内共生体的数量尚不清楚。利用定量聚合酶链反应，我们将确定在蚜虫生长周期中，蚜虫DNA制备中16S和23S rRNA基因的拷贝数。结果将是内共生菌数量的指示。使用免疫学方法，我们将确定几个必要的内共生成分的量。这包括核糖体（核糖体蛋白S1）， RNA聚合酶（β -亚基），sigma因子和GroEL。从这些研究中获得的结果可能用于通过操纵对蚜虫生存至关重要的内共生体来最终控制蚜虫种群。＊＊＊

9402813 Baumann Aphids are dependent for survival on an association with a prokaryotic endosymbiont housed within specialized cells in the aphid body cavity. Using the aphid Schizaphis graminum ( greenbud) we have found that its endosymbiont (Buchnera aphidicola) has all of the genes of the tryptophan biosynthetic pathway and the trpEG (genes for anthranilate synthase, the first enzyme of the pathway) is amplified on a plasmid. These results are consistent with past observations indicating that the endosymbiont synthesizes tryptophan which is utilized by the aphid host. Preliminary results have indicated that, in the distantly related endosymbiont of the aphid Schlectendalia chinensis, tryEG has a chromosomal location. We intend to continue this research by examining the gene organization of the tryptophan biosynthetic pathway in the endosymbiont of this aphid species and making a survey to determine the distribution of gene amplification of chromosomal location of trpEG in the endosymbionts of other aphid species. The first reaction which initiates the biosynthesis of aromatic amino acids (tryptophan, phenylalanine, tyrosine) as well as folic acid is catalyzed by 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. We intend to determine if this gene is located on a plasmid, on the chromosome or if there are isofunctional DAHP synthases. This will be accomplished by restriction enzyme and Southern blot analysis as well as the cloning and sequencing of the gene(s) for the enzyme(s). Comparisons will be made with the gene organization in E. coli and other organisms. In most prokaryotes the genes for ribosomal RNA are arranged as a single transcription unit in the order 16S, and 5S rRNAs. We have shown that B. aphidicola has only one copy of the 16S 23s rRNA gene and is unusual in that it is unlinked to the other rRNA genes. We will continue this study by examining the organization of the 23S and 5S rRNA genes in B. aphidicola. The genes will be cloned, sequenced, and the up stream and downstream regions compared to the putative promoter and terminator of the B.aphidicola 16S rRNA transcription unit. In their most active stage of growth aphids reproduce by parthenogenesis giving birth to live yound. Nothing is known concerning the number of endosymbionts during the growth cycle of the aphid. Using the quantitative polymerase chain reaction we will determine the number of copies of 16S and 23S rRNA genes in the B. aphidicola-aphid DNA preparations during the aphid growth cycle. The results will be an indication of the number of endosymbionts. Using immunological methods we will determine the amount of several essential endosymbiont consitutents. This includes ribosomes (ribosomal protein S1), RNA polymerase (beta-subunit), sigma factor and GroEL. %%% The results obtained from these studies may be of use for the eventual control of aphid populations by manipulation of the endosymbionts which are essential for their survival. ***