Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree.
Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree.
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简单、快速、高效的基因型Nisqually-1转化：首个测序模型树的基本工具
DOI：
10.1038/s41598-017-02651-x
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发表时间：
2017-06-01
影响因子：
4.6
通讯作者：
Cheng Y
中科院分区：
文献类型：
--
作者：
Li S;Zhen C;Xu W;Wang C;Cheng Y
Nisqually-1基因型是第一个具有良好标记基因组的模式木本植物。然而，Nisqually-1的简单而快速的转换仍有待建立。本研究以nisqual -1为材料，采用叶柄和茎段外植体进行再生。外植体切口在2周内形成大量芽。优化后的芽再生培养基（SRM）中含有0.03 mg l - 1苄基氨基嘌呤、0.02 mg l - 1吲哚-3-丁酸和0.0008 mg l - 1噻脲。在此基础上，以含有β-葡萄糖醛酸酶（GUS）为报告基因的载体pBI121为载体，研究了农杆菌介导的茎外植体遗传转化。因此，对影响gus阳性芽转化频率的因素进行优化：OD600为0.4的农杆菌细胞悬液，侵染时间为20 min，共培养时间为2 d，在农杆菌侵染悬液和共培养SRM中加入80µM乙酰丁香酮。利用该方法，在2个月内获得了Nisqually-1转基因植株，平均转化率为26.7%。Southern blot和GUS活性染色证实外源GUS基因已整合到Nisqually-1中。该系统具有快速、高效、操作简单等优点，将进一步提高Nisqually-1在该模型树中的遗传应用。
Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l−1 6-benzylaminopurine, 0.02 mg l−1 indole-3-butyric acid and 0.0008 mg l−1 thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI121 that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD600 of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 – with an average transformation frequency of 26.7% – were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.