Gene Analysis and Cloning for Cellulose-Producing Bacteria : A, Xylinum.

纤维素生产细菌的基因分析和克隆：A，Xylinum。

• 批准号: 01550683
• 负责人: TAKAI Mitsuo
• 金额: $ 1.09万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01550683/
• 关键词: Acetobacter xylinum; Bacterial Cellulose; Plasmid; Shuttle Vector; Transformation; Shotgun Cloning; Restriction Mapping; B-isopropylmalate dehydrogenase gene; ロイシン合成遺伝子; ビオチン; サザンハイブリダイゼ-ション; キメラプラスミド; シャトルベクタ-; 形質転換

Cellulose - producing bacterium Acetobacter aceti subsp. xylinum IFO 3288 was found to harbor seven plasmids named pFF1-7. The pFF plasmids had molecular weights of 1.4 - 9.4 kb and they were characterized with restriction endonucleases. Shuttle vector pUF106 was constructed by ligation of pFF6 to Escherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. This vector transformed cellulose-producing Acetobacter xylinum ATCC 10245 as well as E. coli JM109. Thus shuttle vector pUF106 is available as a practical cloning vector for cellulose- producing bacteria.As a model experiment, the beta -isopropylmalate dehydrogenase (beta -IPMDH) gene, which concerned with Leucine synthesis, of cellulose-producing A. xylinum ATCC 10245 were cloned by shotgun cloning method using leuB-mutant host E. coli C600 and plasmid vector pUC18. Plasmid vector pUL1, carrying a 6.5 kilobase pairs (kb) BamHI-fragment, conferred the beta -IPMDH activity to the wild type of E. coli C600. Restriction mapping and deletion analysis indicated that the beta -IPMDH gene was located on a 1.15kb HindIII-fragment of plasmid pULH1. The cloned beta -IPMDH gene was identified with the original genes of A. Xylinum ATCC 10245 by Southern hybridization method using a biotin-labeled probe.

产纤维素细菌醋酸杆菌醋酸亚种。发现木霉菌IFO 3288含有7个质粒，命名为pFF 1 -7。pFF质粒具有1.4 - 9.4kb的分子量，并且它们用限制性内切酶表征。将pFF 6与大肠杆菌质粒pUC 18连接，构建穿梭载体pUF 106。它具有适合插入外源DNA片段的独特限制性位点，并赋予宿主氨苄青霉素抗性。该载体转化了产纤维素的木醋杆菌ATCC 10245以及E. coli JM 109。作为模型实验，对纤维素产生菌A.木霉菌ATCC 10245的leuB突变宿主E. coliC 600和质粒载体pUC 18。质粒载体pUL 1携带6.5kb的BamH Ⅰ片段，赋予野生型E. coli C600。酶切图谱和缺失分析表明，β-IPMDH基因位于质粒pULH 1的1.15kb的HindIII片段上。克隆的β-IPMDH基因与A.木霉菌ATCC 10245通过Southern杂交方法使用生物素标记的探针。

项目成果

高井 光男: "Filtration and Permeation Characteristics of Bacterial Cellulose Composite" 繊維学会誌. 47. (1991)

Mitsuo Takai：“细菌纤维素复合物的过滤和渗透特性”日本纺织技术研究所杂志47。（1991）

藤原 政司: "Construction of Shuttle Vectors and A Genetic Transformation System for CelluloseーProducing Bacteria Acetobader Xylinum" Cellulose; Structural and functional aspects (Ellis Horwood Publishers). 153-158 (1989)

Masashi Fujiwara：“纤维素穿梭载体和遗传转化系统的构建——木醋巴菌生产菌”纤维素；结构和功能方面（Ellis Horwood Publishers）。

Y. Shimizu, J. Hayashi: "Acetylation of Cellulose with Carboxylic Acids" Cellulose Chem. Technol.23. 661-670 (1989)

Y. Shimizu、J. Hayashi：“纤维素与羧酸的乙酰化”纤维素化学。

高井 光男: "分解性プラスチック(分担執筆,監修:土肥義治)“バイオセルロ-ス"" CMC(シ-エムシ-), 276 (1990)

高井光男：“可降解塑料（撰稿人、监督者：土井义晴）‘生物纤维素’”CMC，276 (1990)

藤原政司: "Cellulose:structural and functional aspects" Ellis Horwood,(London), 5 (1990)

Masashi Fujiwara：“纤维素：结构和功能方面”Ellis Horwood，（伦敦），5 (1990)