For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.

译文

首次将磷酸甘露糖异构酶 (PMI,EC 5.3.1.8)/基于甘露糖的 “阳性” 选择系统用于获得基因工程甘蔗 (Saccharum spp. hybrid var. CP72-2086) 植物。以不可翻译的甘蔗花叶病毒 (SCMV) 菌株E外壳蛋白 (CP) 基因和大肠杆菌PMI基因manA作为选择标记基因,对胚性愈伤组织进行生物形态转化后,获得了甘蔗的转基因品系。轰击后,转基因愈伤组织在含有13.6 microM 2,4-d,20g l(-1) 蔗糖和3g l(-1) 甘露糖的修饰的MS培养基上选择性增殖。在相似的选择条件下,在具有2.5 microM TDZ的MS基础培养基上获得植物再生,并且再生剂在具有19.7 microM IBA,20g l(-1) 蔗糖和1.5g l(-1) 甘露糖的MS基础培养基上生根。3周后,甘露糖浓度从允许 (1.5g l(-1)) 增加到选择性 (3g l(-1)) 条件,通过减少选择逃逸的数量,提高了整体转化效率。34株生长旺盛的推定转基因植物已成功移植到温室中。PCR和Southern印迹分析表明,有19株植物为manA阳性,有15株植物为CP阳性,而13种独立的转基因包含两种转基因。使用氯酚红测定和酶促分析评估了转基因植物中法力的表达。

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