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

半胱氨酸合成酶(O-乙酰丝氨酸(硫醇)-裂解酶，EC 4.2.99.8)催化植物硫同化的关键步骤--L-半胱氨酸由O-乙酰丝氨酸和硫化氢生成。本文报道了菠菜半胱氨酸合成酶基因克隆的分离和鉴定。从纯化的CSase的V8酶切片段中获得内源多肽序列。以菠菜幼绿叶的Poly(A)^+RNA为模板，构建了lambdagt10基因文库。用合成的两个编码部分肽序列的混合核苷酸进行筛选，从2×10~(-5)个克隆中筛选出19个阳性杂交克隆。对两个相互独立的克隆的核苷酸序列分析表明，该克隆有一个连续的开放阅读框，编码325个氨基酸的多肽，计算的相对分子质量为34,185 Da。推导的氨基酸序列与大肠杆菌和鼠伤寒沙门氏菌CSase的同源性为53%。序列同源性还观察到与细菌和酵母中其他氨基酸代谢酶以及大鼠血红蛋白H-450的同源性。构建了一个细菌表达载体，该表达载体能够在基因上补充大肠杆菌营养缺陷型CSase。Western blotting和酶活性测定也证明了菠菜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.