Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems
Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems
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DOI：
10.1006/abbi.2000.1867
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发表时间：
2000-07-01
影响因子：
3.9
通讯作者：
Werck-Reichhart, D
中科院分区：
文献类型：
--
作者：
Batard, Y;Hehn, A;Werck-Reichhart, D
单子叶作物通常比禾本科杂草和大多数双子叶植物更耐除草剂。它们对除草剂的抗性在许多情况下由P450加氧酶介导。因此，单子叶植物构成了一个有吸引力的来源P450酶操纵除草剂抗性和重组形式的主要异生物质代谢mooxygenases是潜在的工具，优化新的活性分子。我们在这里报告的分离和功能特性的第一个P450和P450还原酶编码序列从小麦，在酵母和烟草中表达这些cDNA的第一次尝试导致蛋白质的水平，这仅仅是非常低的，往往甚至无法检测。小麦P450 cDNA被高效转录，但没有蛋白质或活性的found.Wheat编码序列，像那些其他单子叶植物，其特点是由一个高GC含量和相关的强偏好的密码子使用，从酵母或双子叶植物中观察到的不同。基因的完全重新编码是昂贵的，尝试使用设计成符合宿主密码子使用的单个PCR大引物来重新工程化它们的5 '端。这足以缓解翻译抑制并获得良好的蛋白质表达水平。同样的策略也导致了烟草中蛋白质表达的急剧增加。这样的部分重编码策略，更容易和更便宜的cDNA的完全重编码的成功的基础，提出。(C)北京大学出版社.
Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat, The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which mere extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed. (C) 2000 Academic Press.