Transgenic Research ( IF 2.7 ) Pub Date : 2021-05-06 , DOI: 10.1007/s11248-021-00255-w M E González-Romero 1, 2 , C Rivera 1, 3 , K Cancino 1, 4 , F Geu-Flores 5 , E G Cosio 6 , M Ghislain 1 , B A Halkier 2
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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.
Graphical abstract
中文翻译：
对马铃薯植物进行生物工程改造，生产苄基芥子油苷，以提高广谱病虫害抗性
在安第斯山脉的传统小规模农业中，马铃薯经常与安第斯食用块茎Tropaeolum tuberosum（俗称 mashua）共同种植，由于其含有苯丙氨酸，人们相信马铃薯具有防治病虫害的作用。苄基芥子油苷（BGLS）。我们通过使用两个多顺反子构建体连续产生稳定的转基因事件，对马铃薯中 BGLS 的生产进行了生物工程，所述多顺反子构建体编码来自拟南芥的六个 BGLS 生物合成基因的表达。首先，我们将编码该途径最后三个基因（ 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，我们堆叠了第二个多顺反子构建体，编码该途径中的前三个基因（ CYP79A2、CYP83B1和GGP1 ），由来自菊花的rubisco小亚基的叶特异性启动子驱动。与产生不可检测水平的 BGLS 的非转基因马铃薯植物相比，我们获得了产生高达 5.18 pmol BGLS/mg 鲜重的转基因事件。初步生物测定表明可能具有抗致病疫霉（引起晚疫病）和Premnotrypes suturicallus （称为安第斯马铃薯象甲虫）的活性。然而，我们在十个选定的转基因事件中有五个观察到叶子形态改变、叶子异常厚且卷曲、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具有不良影响。 需要优化马铃薯中 BGLS 生物合成途径的表达，以避免植物发育的改变。
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