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来自大肠杆菌的dsdA基因可为植物转化提供一种新型选择标记。

The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation.

作者信息

Erikson Oskar, Hertzberg Magnus, Näsholm Torgny

机构信息

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå.

出版信息

Plant Mol Biol. 2005 Feb;57(3):425-33. doi: 10.1007/s11103-004-7902-9.

Abstract

Plants are sensitive to D-serine, but functional expression of the dsdA gene, encoding D-serine ammonia lyase, from Escherichia coli can alleviate this toxicity. Plants, in contrast to many other organisms, lack the common pathway for oxidative deamination of D-amino acids. This difference in metabolism has major consequences for plant responses to D-amino acids, since several D-amino acids are toxic to plants even at relatively low concentrations. Therefore, introducing an enzyme specific for a phytotoxic D-amino acid should generate a selectable characteristic that can be screened. Here we present the use of the dsdA gene as a selectable marker for transformation of Arabidopsis. D-serine ammonia lyase catalyses the deamination of D-serine into pyruvate, water and ammonium. dsdA transgenic seedlings can be clearly distinguished from wild type, having an unambiguous phenotype immediately following germination when selected on D-serine containing medium. The dsdA marker allows flexibility in application of the selective agent: it can be applied in sterile plates, in foliar sprays or in liquid culture. Selection with D-serine resistance was compared with selection based on kanamycin resistance, and was found to generate similar transformation frequencies but also to be more unambiguous, more rapid and more versatile with respect to the way the selective agent can be supplied.

摘要

植物对D-丝氨酸敏感,但来自大肠杆菌的编码D-丝氨酸氨裂解酶的dsdA基因的功能表达可以减轻这种毒性。与许多其他生物不同,植物缺乏D-氨基酸氧化脱氨的常见途径。这种代谢差异对植物对D-氨基酸的反应有重大影响,因为即使在相对较低的浓度下,几种D-氨基酸对植物也是有毒的。因此,引入一种对具有植物毒性的D-氨基酸具有特异性的酶应该会产生一种可筛选的选择特性。在这里,我们展示了使用dsdA基因作为拟南芥转化的选择标记。D-丝氨酸氨裂解酶催化D-丝氨酸脱氨生成丙酮酸、水和铵。dsdA转基因幼苗可以与野生型明显区分开来,在含有D-丝氨酸的培养基上选择时,发芽后立即具有明确的表型。dsdA标记在选择剂的应用上具有灵活性:它可以应用于无菌平板、叶面喷雾或液体培养中。将基于D-丝氨酸抗性的选择与基于卡那霉素抗性的选择进行了比较,发现二者产生的转化频率相似,但在选择剂的供应方式方面,基于D-丝氨酸抗性的选择更明确、更快速且更通用。

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