ISOLATION OF AN ARABIDOPSIS-THALIANA GENE ENCODING CYCLOARTENOL SYNTHASE BY FUNCTIONAL EXPRESSION IN A YEAST MUTANT LACKING LANOSTEROL SYNTHASE BY THE USE OF A CHROMATOGRAPHIC SCREEN
ISOLATION OF AN ARABIDOPSIS-THALIANA GENE ENCODING CYCLOARTENOL SYNTHASE BY FUNCTIONAL EXPRESSION IN A YEAST MUTANT LACKING LANOSTEROL SYNTHASE BY THE USE OF A CHROMATOGRAPHIC SCREEN
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DOI：
10.1073/pnas.90.24.11628
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发表时间：
1993-12-15
影响因子：
11.1
通讯作者：
BARTEL, B
中科院分区：
文献类型：
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作者：
COREY, EJ;MATSUDA, SPT;BARTEL, B
脊椎动物和真菌通过中间体羊毛甾醇从环氧角鲨烯合成甾醇，而植物将环氧角鲨烯环化为环阿屯醇作为初始甾醇。我们报道了拟南芥（Arabidopsis thaliana）中编码环阿屯醇合酶（cycloartenol synthase）的基因CAS 1的克隆和特性分析[（S）-2，3-epoxysqualene（cyclozing，cycloartenol forming），EC 5.4.99.8]。将缺乏羊毛甾醇合酶的酵母突变体[（S）-2，3-环氧角鲨烯（环化，羊毛甾醇形成），EC 5.4.99.7]用A. thaliana cDNA酵母表达文库，并通过薄层色谱法测定菌落的环氧-角鲨烯降解活性。几乎等于10，000个转化体中的一个产生了将2，3-环氧角鲨烯环化为植物甾醇环阿屯醇的匀浆。该活性显示为质粒依赖性的。质粒插入物含有2277-bp的开放阅读框，其能够编码与来自白色念珠菌的羊毛甾醇合酶和角鲨烯-霍烯环化酶（EC 5.4.99.-）具有显著同源性的86-kDa蛋白。来自酸钙芽孢杆菌用于克隆该基因的方法应该普遍适用于负责次级代谢物生物合成的基因。
Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxy-squalene mutase activity by thin-layer chromatography. One out of almost-equal-to 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.