Application of the phosphomannose-isomerase/mannose selection system in the Agrobacterium-mediated transformation of Lonicera hypoglauca Miq.
Application of the phosphomannose-isomerase/mannose selection system in the Agrobacterium-mediated transformation of Lonicera hypoglauca Miq.
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DOI：
10.1007/s13562-020-00570-z
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发表时间：
2020-05-24
影响因子：
1.9
通讯作者：
Jiang, Ni
中科院分区：
文献类型：
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作者：
Shi, Fenghua;Tan, Muxiu;Jiang, Ni
红蓝紫枝兰的干燥芽。具有解热、解毒、抗炎的作用，金银花被广泛用于中成药中。由于其药用价值，有必要建立其转化系统，以研究其活性物质的生物合成途径。磷酸甘露糖异构酶（PMI）基因可能是植物转化系统中最有效的选择标记。建立转化番茄的PMI筛选体系。以昆明山海棠为试材，在附加不同比例甘露糖和蔗糖的诱导培养基（MS +0.1mg/LNAA +1.0mg/L 6-BA + 6 g/L琼脂）上培养，收集愈伤组织形成的数据。在确定甘露糖和蔗糖浓度后，用含pCAMBIA 1301-PMI的农杆菌感染172个叶片外植体、61个叶柄和53个茎段外植体。用PCR、RT-PCR和氯酚红法对诱导的愈伤组织进行检测。我们发现，愈伤组织的形成有效地抑制诱导培养基上补充甘露糖。选择培养基中适宜的甘露糖和蔗糖浓度分别为8 g/L和22 g/L，在此浓度下，L.苍白占15.96%。以RT-PCR为标准，对叶柄和茎段诱导的愈伤组织进行CPR检测有一定的假阴性率。因此，PMI/甘露糖选择系统可用于L. CPR法适合于转化L. hypoglauca的初步筛选。hypoglauca。
The dried buds of Lonicera hypoglauca Miq. have antipyretic, antidotal and anti-inflammatory properties and as Flos lonicerae are widely used in Chinese patent medicine. Because of its medicinal value, there is a need to establish its transformation system in order to study the biosynthesis pathway of its active substances. The phosphomannose isomerase (PMI) gene is probably the most effective selection maker for the transformation system of plants. To establish PMI selection system for transformation of L. hypoglauca, explants were cultured on the induction medium (MS + 0.1 mg/L NAA + 1.0 mg/L 6-BA + 6 g/L agar) supplemented with different ratios of mannose and sucrose, and data on callus formation were collected. After the concentration of mannose and sucrose was determined, 172 leaf explants, 61 leafstalks and 53 stem segment explants were infected with Agrobactrium containing pCAMBIA1301-PMI. The putatively transformed calluses induced from these explants were detected by PCR, RT-PCR and chlorophenol red assay (CPR). We found that callus formation was effectively suppressed on the induction medium supplemented with mannose. The appropriate concentration of mannose and sucrose in the selection medium was 8 g/L and 22 g/L, under which the transformation frequency of leaf from L. hypoglauca was 15.96%. CPR assay for callus induced from leafstalk and stem have a moderate false negative rate when RT-PCR used as criterion. Taken together, it was concluded that PMI/mannose selection system could be used in the transformation of L. hypoglauca, and CPR assay might be suited for the preliminary screening of the transformation of L. hypoglauca.