Geu-Flores Fernando, Olsen Carl Erik, Halkier Barbara Ann
Plant Biochemistry Laboratory, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, 40 Thorvaldsensvej, 1871, Frederiksberg C, Denmark.
Planta. 2009 Jan;229(2):261-70. doi: 10.1007/s00425-008-0825-y. Epub 2008 Oct 2.
Glucosinolates are amino acid-derived secondary metabolites present in cruciferous plants. Glucosinolates and their hydrolysis products are involved in defence against insects and pathogens, but are also known for their characteristic flavor and their cancer-preventive and antibacterial properties. This wide range of bioactivities has prompted a desire to engineer glucosinolates into non-cruciferous plants. We report the one-step transfer of the last three steps of the benzylglucosinolate pathway (comprising the C-S lyase, glycosyltransferase and sulfotransferase) from Arabidopsis to tobacco. This was achieved using an expression construct consisting of a single 2A polycistronic open reading frame, which allowed the expression of the three coding-sequences from a single promoter. When compared to wildtype plants, transgenic tobacco lines showed increased ability to convert the intermediate phenylacetothiohydroxamate to benzylglucosinolate upon in vivo feeding. Enzymatic assays using plant extracts demonstrated that the individual activities required for this conversion were enhanced in the transgenic plants. The relatively high conversion by wildtype plants in feeding assays supports the hypothesis that the last part of the glucosinolate pathway was recruited from existing detoxification reactions. Immunoblots confirmed that individual proteins were being successfully produced from the 2A polycistronic open reading frame, albeit fusion proteins could also be detected. In summary, we transferred the last three steps of the benzylglucosinolate pathway to tobacco as a first step towards engineering glucosinolates into non-cruciferous plants.
硫代葡萄糖苷是十字花科植物中由氨基酸衍生而来的次生代谢产物。硫代葡萄糖苷及其水解产物参与植物对昆虫和病原体的防御，同时也因其独特的风味、防癌和抗菌特性而闻名。这种广泛的生物活性促使人们希望将硫代葡萄糖苷导入非十字花科植物中。我们报道了将苄基硫代葡萄糖苷途径的最后三个步骤（包括C-S裂解酶、糖基转移酶和磺基转移酶）从拟南芥一步转移到烟草中的方法。这是通过使用一个由单个2A多顺反子开放阅读框组成的表达构建体实现的，该构建体允许从单个启动子表达三个编码序列。与野生型植物相比，转基因烟草品系在体内喂食时将中间产物苯乙酰硫代羟肟酸转化为苄基硫代葡萄糖苷的能力增强。使用植物提取物进行的酶活性测定表明，转基因植物中这种转化所需的各个活性均有所增强。野生型植物在喂食试验中的相对高转化率支持了硫代葡萄糖苷途径的最后一部分是从现有的解毒反应中招募而来的这一假设。免疫印迹证实，尽管也能检测到融合蛋白，但单个蛋白质是从2A多顺反子开放阅读框中成功产生的。总之，我们将苄基硫代葡萄糖苷途径的最后三个步骤转移到了烟草中，这是将硫代葡萄糖苷导入非十字花科植物的第一步。
