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GENES STRUCTURES, EXPRESSIONS, REGULATIONS AND MECHANISMS OF EXCREATION OF PECTINOLYTIC ENZYMES

果胶分解酶分泌的基因结构、表达、调控和机制

基本信息

批准号：
03453135
负责人：
IZAKI Kazuo
金额：
$ 4.22万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Genes structures of pectate lyase and pectin lyase of Erwinia carotovora Er, their expression and regulation and excretion of pectate lyases from the cells were studied in this research. Firstly, pectate lyase gene II was cloned and the complete nucleotides of pectate lyase gene II were sequenced in addition to pectate lyase genes I and III which were already determined. Complete nucleotides of pectate lyase I were also sequenced and some sequences were revised. Pectate lyase genes I.II.III were found to be closely and tandemly lined as a cluster. Secondly, the complete nucleotides of pectin lyase gene were sequenced, and found to be composed of 942 bp. From this result, pectin lyase was considered to be a protein of molecular mass of 33,700 composed of 314 amino acid residues. Regions of -35, -30, SD sequence and two parindromic regions were found in the 5'upstream of pectin lyase. However, Lex A binding site which is found in operator region of SOS regulon of E.coli. From the experiment using the deletion mutants in the 5'upstream region of pectin lyase, expression of pectin lyase genes seemed to be not controlled by repressor as in SOS reaction. Thirdly, pectate lyases I,II,III were not excreated from the cells of E.coli, while they were excreated efficiently from the cells of Erwinia carotovora Er. Further experiments are necessary to explain this difference.

本研究对胡萝卜软腐欧文氏菌（Erwinia carotovora Er）果胶酸裂解酶和果胶裂解酶的基因结构、表达调控以及果胶酸裂解酶的分泌进行了研究。首先，克隆了果胶酸裂解酶基因II，并测定了果胶酸裂解酶基因II的全核苷酸序列以及已测定的果胶酸裂解酶基因I和III。对果胶酸裂解酶Ⅰ的全基因组核苷酸序列进行了测定，并对部分序列进行了修正。果胶酸裂解酶基因I、II、III紧密串联成簇。其次，对果胶裂解酶基因进行了全序列测定，发现其全长942 bp。根据该结果，认为果胶裂解酶是由314个氨基酸残基组成的分子量为33，700的蛋白质。在果胶裂解酶的5 '端上游存在-35、-30、SD序列区和两个平行序列区。然而，在大肠杆菌SOS调节子操纵子区域发现的Lex A结合位点。从使用果胶裂解酶5 '上游区域的缺失突变体的实验来看，果胶裂解酶基因的表达似乎不像SOS反应中那样受阻遏物控制。第三，果胶酸裂解酶I、II、III不能从大肠杆菌细胞中表达，而能从胡萝卜软腐欧文氏菌细胞中有效表达。需要进一步的实验来解释这种差异。