Cloning and expression of genes responsible for secondary metabolism in medicinal plants.

负责药用植物次生代谢的基因的克隆和表达。

基本信息

批准号：
02807199
负责人：
SAITO Kazuki
金额：
$ 1.09万
依托单位：
Chiba 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：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Cysteine synthase(CSase)(O-acetylserine(thiol)-lyase, EC 4.2.99.8)catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here isolation and characterization of cDNA clones encoding cysteine synthase from spinach(Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambdagt10 cDNA library was constructed from poly(A)^+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences gave 19 positively hybridized clones from 2x10^5 clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34, 185 Da. Sequence comparison of deduced amino acids showed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes of amino acids in bacteria and yeast, and rat hemoprotein H-450. A bacterial expression vector was constructed and this plasmid was able to genetically complement an E. coli auxotroph deficient CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by Western blotting and the assay of enzymatic activity.

半胱氨酸合成酶 (CSase)（O-乙酰丝氨酸（硫醇）-裂合酶，EC 4.2.99.8）催化 O-乙酰基-L-丝氨酸和硫化氢形成 L-半胱氨酸，这是植物中硫同化的关键步骤。我们在此报告编码菠菜（Spinacia oleracea L.）半胱氨酸合酶的 cDNA 克隆的分离和表征。从纯化的 CSase 的 V8 蛋白酶消化片段中获得内部肽序列。从菠菜嫩绿叶的poly(A)^+ RNA 构建了lambdagt10 cDNA 文库。用编码部分肽序列的两种合成混合核苷酸进行筛选，从 2x10^5 克隆中得到 19 个阳性杂交克隆。对两个独立 cDNA 克隆的核苷酸序列分析揭示了编码 325 个氨基酸的多肽的连续开放阅读框，计算分子量为 34, 185 Da。推导的氨基酸序列比对显示，与大肠杆菌和鼠伤寒沙门氏菌的CSase有53%的一致性。还观察到与细菌和酵母中的其他氨基酸代谢酶以及大鼠血红素蛋白 H-450 的序列同源性。构建了细菌表达载体，并且该质粒能够在遗传上补充大肠杆菌营养缺陷型缺陷型CSase。蛋白质印迹和酶活性测定也证明了大肠杆菌中具有功能活性的菠菜 CSase 的积累。