MOLECULAR CELL BIOLOGICAL STUDIES ON INTRACELLULAR SYMBIOSIS

本研究对棉铃虫胞内共生菌Buchnera和褐飞虱胞内酵母型共生菌Buchnera进行了研究，主要结果如下：1.比较了幼龄和老年棉铃虫细菌体共生系统的基因表达谱，发现寄主细胞和Buchnera都有10多个基因只有在共生系统保持功能的情况下才能特异表达。这些基因包括参与氨基酸新陈代谢的酶的基因，证实了我们之前的结果。我们获得了令人信服的证据，表明Buchnera产生核黄素并向宿主提供维生素。我们用限制性内切酶消化含有丰富G/C酶切位点的基因组DNA，然后用脉冲场凝胶电泳法估计了Buchnera的基因组大小。结果发现，其基因组大小为657kbp，仅为E.coli.4基因组的七分之一。对两个不同基因的斑点杂交分析和细胞DNA含量的直接荧光分析表明，Buchnera是非常特殊的细菌，单个细胞中有100多个基因组拷贝。此外，由于寄主昆虫的形态和发育阶段不同，其基因组拷贝数也存在很大的差异。作为细菌，Buchnera是例外的，因为它们含有一种类似组蛋白Hi的蛋白质，这种蛋白质可能与基因组DNA相互作用。我们成功地克隆了类酵母共生体的尿酸酶基因，这是稻飞虱动员组织中储存的尿酸所必需的。此外，我们还构建了尿酸酶基因的大规模表达系统，将尿酸酶基因连接到表达载体中，转化E.

We studied on Buchnera, intracellular symbiotic bacteria of aphids and intracellular yeast-like symbionts of brown planthoppers, the results of which are summarized as follows :1. Comparison of gene expression patterns of the bacteriocyte symbiotic systems from young and old aphids revealed that there are more than ten genes, of both host cell and Buchnera, that are specifically expressed only when the symbiotic system is kept functional. These genes include those for enzymes involved in the amino acid metabolism, confirming our previous results.2. We attained convincing evidence that Buchnera produce riboflavin and provision the vitamin to hosts.3. We estimated the genome size of Buchnera by digesting the genomic DNA with restriction enzymes whose restriction sites are rich in G/C, followed by pulse-field gel electrophoresis. It turned out that the genome size, 657kbp, is only one seventh of that of the genome of E.coli.4. Dot-blot analyses of two different genes and direct fluorimetry of the cellular DNA content revealed that Buchnera are very extraordinary bacteria with more than 100 genomic copies in a single cell. In addition, the genomic copy number of Buchnera is variable to a great extent due to morph and developmental stage of the host insect.5. Buchnera are exceptional as bacteria in that they contain a histone Hi -like protein, which is likely in interaction with the genomic DNA.6. We successfully cloned the gene for uricase of the yeast-like symbiont that is essential for planthoppers to mobilize the uric acid stored in their tissue. Also, we constructed a large scale expression system of the uricase gene by transforming E.coil with the gene ligated into an expression vector.