Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants
Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants
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木豆高效根转基因系统的开发及其在其他重要经济植物中的应用
DOI：
10.1111/pbi.13101
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发表时间：
2019-09-01
影响因子：
13.8
通讯作者：
Fu, Yujie
中科院分区：
文献类型：
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作者：
Meng, Dong;Yang, Qing;Fu, Yujie
对于非模式植物，由于缺乏有效的稳定的转化程序，基因的功能鉴定仍然受到阻碍。在这里，我们报道了一种简单、快速、高效的发根农杆菌转化技术，可以在活体植物中产生稳定的转基因根，为体内功能研究提供便利。我们发现，将发根农杆菌注射到不同植物的茎中可以产生稳定的转基因根，在切断原始的非转基因根后，可以维持植物的生长。通过对木本主要粮食作物木豆的菌种筛选、菌种浓度、注射部位和苗龄的优化，建立了木豆遗传转化体系。RT-PCR和荧光观察表明，转基因木豆的生根率约为39%。此外，在测试的12株重要经济植物中，有9株获得了毛状根的诱导，效率为15%-39%。作为概念验证，应用双分子荧光互补(BIFC)分析了木豆中CcCIPK14和CcCBL1/2之间的相互作用。此外，bZIP转录因子MdHY5在苹果中的异源表达证实了该转化技术在根中工程合成花青素方面的实用性。综上所述，我们表明，这种方法可以在广泛的植物物种中快速进行基因功能的体内研究。
For non-model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non-transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT-PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15-39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.