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

半胱氨酸合成酶（CSase）（o -乙酰丝氨酸（硫醇）裂解酶，EC 4.2.99.8）催化o -乙酰-l -丝氨酸和硫化氢形成l-半胱氨酸，是植物硫同化的关键步骤。本文报道了从菠菜（Spinacia oleracea L.）中分离和鉴定编码半胱氨酸合成酶的cDNA克隆。从纯化的CSase的V8蛋白酶酶切片段中获得内部肽序列。利用菠菜嫩叶的poly(A)^+ RNA构建了lambdagt10 cDNA文库。用编码部分肽序列的两个合成混合核苷酸进行筛选，从2 × 10^5个克隆中获得19个阳性杂交克隆。对两个独立的cDNA克隆进行核苷酸序列分析，发现一个连续开放阅读框编码325个氨基酸的多肽，计算分子质量为34,185 Da。与大肠杆菌和鼠伤寒沙门菌的氨基酸序列比较，其同源性为53%。与细菌和酵母菌中氨基酸的其他代谢酶以及大鼠血红蛋白H-450也有序列同源性。构建了一种细菌表达载体，该质粒能够对大肠杆菌营养缺陷病例进行基因补充。Western blotting和酶活性测定也证实了功能活性菠菜CSase在大肠杆菌中的积累。

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.