Applied Biotechnology Laboratory, International Potato Centre, P.O. Box 1558, Lima, 12, Peru.
Department of Plant and Environmental Sciences, DynaMo Center, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark.
Transgenic Res. 2021 Oct;30(5):649-660. doi: 10.1007/s11248-021-00255-w. Epub 2021 May 6.
In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development.
在安第斯山脉传统的小规模农业中，马铃薯常与安第斯可食用块茎颠茄茄（俗称马舒阿）共同种植，颠茄茄因其含有苯丙氨酸衍生的苄基葡萄糖苷（BGLS）而被认为具有防治病虫害的作用。我们通过连续几代稳定的转基因事件，在马铃薯中生物工程生产 BGLS，这些事件使用了来自拟南芥的六个 BGLS 生物合成基因的两个多顺反子构建体进行表达。首先，我们将一个编码途径中后三个基因（SUR1、UGT74B1 和 SOT16）的多顺反子构建体整合到由花椰菜花叶病毒 35S 启动子驱动的马铃薯中。在鉴定出具有最高转基因表达的单插入转基因事件后，我们在由菊花 Rubisco 小亚基叶片特异性启动子驱动的第二个多顺反子构建体中堆叠了编码途径中前三个基因（CYP79A2、CYP83B1 和 GGP1）的多顺反子构建体。我们获得的转基因事件产生的 BGLS 比非转基因马铃薯植物高 5.18 pmol/mg 鲜重，而后者几乎检测不到 BGLS。初步生物测定表明，它可能对引起晚疫病的致病疫霉和被称为安第斯马铃薯象鼻虫的 Premnotrypes suturicallus 具有活性。然而，我们观察到在十个选定的转基因事件中有五个表现出叶片形态改变、叶片异常增厚卷曲、生长和块茎产量减少，这表明 BGLS 生物合成基因的表达对马铃薯有不良影响。需要优化 BGLS 生物合成途径在马铃薯中的表达，以避免改变植物的发育。
