Symbiotic Association of Aphids: Biochemical Role of the Prokaryotic Endosymbiont

蚜虫的共生关联：原核内共生体的生化作用

• 批准号: 9402813
• 负责人: Paul Baumann
• 金额: $ 31万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9402813&HistoricalAwards=false
• 关键词: Symbiotic; Association; Aphids; Biochemical; Role

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. ***

9402813鲍曼蚜虫的生存依赖于与原核内共生菌的联系，这种共生菌存在于蚜虫体腔的特殊细胞中。利用麦长管蚜Schizaphim graminum(Greenbud)，我们发现其内共生菌(Buchnera Aphidicola)具有色氨酸生物合成途径的所有基因，并在一个质粒上扩增出trpEG(邻氨基苯甲酸合成酶基因，该途径的第一种酶)。这些结果与过去的观察结果一致，表明内生共生菌合成色氨酸，而色氨酸被蚜虫寄主利用。初步研究结果表明，在与之有较远亲缘关系的麦长管蚜的内生共生体中，triEG具有染色体定位。我们打算通过检测该种类内共生体中色氨酸生物合成途径的基因组织来继续这项研究，并调查确定trpEG染色体定位的基因扩增在其他种类内共生体中的分布。第一个反应是由3-脱氧-D-阿拉伯-7-磷酸(DAHP)合成酶催化合成芳香族氨基酸(色氨酸、苯丙氨酸、酪氨酸)和叶酸。我们打算确定该基因是否位于质粒、染色体上，或者是否存在相同功能的DAHP合成酶。这将通过限制性内切酶和Southern印迹分析以及酶(S)基因(S)的克隆和测序来完成。将与大肠杆菌和其他生物中的基因组织进行比较。在大多数原核生物中，核糖体RNA的基因以16S和5S rRNAs的顺序排列为单个转录单位。我们已经证明了B.aphidicola只有一个16S 23S rRNA基因的拷贝，而且不寻常的是它与其他rRNA基因没有连锁。我们将通过检测B.aphidicola中23S和5S rRNA基因的组织来继续这项研究。这些基因将被克隆、测序，并将上游和下游区域与B.aphidicola 16S rRNA转录单位的假定启动子和终止子进行比较。在其最活跃的生长阶段，蚜虫通过孤雌生殖繁殖，并在附近生活。关于蚜虫生长周期中内共生菌的数量，目前尚不清楚。利用定量聚合酶链式反应，我们将测定在棉铃虫生长周期中，棉铃虫DNA样品中16S和23S rRNA基因的拷贝数。这一结果将是内共生菌数量的一个指示。使用免疫学方法，我们将测定几种必需的内共生菌成分的数量。这包括核糖体(核糖体蛋白S1)、RNA聚合酶(β亚基)、Sigma因子和GroEL。%这些研究结果可能有助于通过控制内共生菌来最终控制蚜虫种群，内共生菌对它们的生存至关重要。***