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题名: 防治同翅目農作物蟲害:以基因組學方法進行同翅目昆蟲內共生菌關鍵基因的分子分析(1/2)
Control of Homopteran Pests: Molecular Analysis of Functionally Significant Proteins from Their Endosymbionts by Genomic Approaches
作者: 賴吉永;溫育德;郭美華;楊曼妙;李奇英
贡献者: 生物系
关键词: 蚜蟲;內共生菌;胺基酸合成酵素;隨護蛋白質
Buchnera;Aphids;Amino acid synthesis;GroE
日期: 2004
上传时间: 2012-07-03T04:03:26Z
出版者: 行政院國家科學委員會
摘要: 蚜蟲是農作物的重要害蟲之一﹐它們的生長繁殖有賴於體內內共生菌 Buchnera 的生化合成
作用﹐本計劃將利用目標導向的藥物開發策略 (targetbased drug discovery)﹐由Buchnera 基
因組中尋找適當的藥物作用目 標基因﹐再利用大腸桿菌表現的基因產物建立細胞外的高通
量篩選平台(high throughput screening platforms)﹐篩選可抑制Buchnera 代謝功能的合成或天
然物質作為發展抗蟲藥物的基礎。計畫第一年內容為選殖Buchnera之groESL隨護蛋白質基
因,以及trpE、cysE、pheA、ilvIH、hisC與serC等胺基酸合成酵素基因,並在大腸桿菌細胞
內表現上述基因。主要目標為藉由groESL的共同表現增加Buchnera酵素基因在大腸桿菌細胞
內的穩定性。並將建構大腸桿菌上述基因的突變株,藉由基因功能互補試驗以及酵素活性
測定檢查基因表現程度,並測定酵素受各別代謝途徑終產物的抑制程度。其中trpE、cysE、
hisC與pheA可補償大腸桿菌相對應之酵素基因突變,共同表現groESL可增進trpE、cysE與
pheA的功能,表現Buchnera pheA的大腸桿菌株具有分泌phenylalanine的特性,顯示此酵素不
受其生化合成途徑終產物phenylalanine之抑制。
Aphids are important agricultural pests. Survival of aphids depends upon the full biosynthetic
capacity of their endosymbiotic bacterium Buchnera aphidicola. We proposed to develop new
aphid controlling pesticides employing a target-based drug discovery strategy: Buchnera genes
suitable as drug targets will be identified from the recently completed Buchnera genome
sequences. These genes will be expressed in E. coli and their products purified for setting up
cell-free enzyme assays. High-throughput drug screening systems based on these assays will then
be used to screen libraries of synthetic and natural compounds for enzyme inhibitors with the
potential to be chemically modified into new aphid controlling agents. The objectives of the first
year include cloning of amino acid biosynthetic genes trpE, cysE, ilvIH, pheA, hisC, serC, and
chaperone genes groESL from the genome of pea aphid primary endosymbiont, followed by
expression of these genes in E. coli and characterization of the enzymatic functions of the gene
products. Co-expression of groESL with enzyme genes was expected to improve stability,
solubility and activity of the enzymes. E. coli strains carrying mutations in these genes were also
constructed and used in gene function complementation experiments to confirm isofunctionality
of these genes with their E. coli homologues. We found that Buchnera trpE, cysE, hisC and pheA
were capable of complementing null mutations of corresponding genes in E. coli. And
co-expression of groESL improved the activities of trpE, cysE and pheA. The possibility that
Buchnera PheA is resistant to feedback inhibition by phenylalanine is suggested by the finding
that E. coli strains carrying Buchnera pheA actively secretes phenylalanine into the culture
medium.
關聯: 國科會計畫, 計畫編號: NSC93-2317-B018-001; 研究期間: 9308-9407
显示于类别:[生物學系] 國科會計畫

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