Germline transformation of Shepherd's purse (Capsella bursa-pastoris) by the 'floral dip' method as a tool for evolutionary and developmental biology
Germline transformation of Shepherd's purse (Capsella bursa-pastoris) by the 'floral dip' method as a tool for evolutionary and developmental biology
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DOI：
10.1016/j.gene.2007.10.033
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发表时间：
2008-02-15
期刊：
影响因子：
3.5
通讯作者：
Theissen, Gunter
中科院分区：
文献类型：
--
作者：
Bartholmes, Conny;Nutt, Pia;Theissen, Gunter
Capsella bursa-Pastoris是进化和发育生物学的一个有吸引力的模式系统。为了便于进一步的基因功能研究，采用花浸法实现了法氏囊-巴斯德乳杆菌的种系转化。分别以GFP和BASTA抗性(Bar)基因为标记筛选法氏囊-巴斯德转基因植株，并利用GUS基因和GFP基因监测转基因植株的表达水平。我们测试了两株农杆菌菌株LBA4404和GV3101转化法氏囊-巴斯德乳杆菌的能力。与两个菌株都能转化不同生态型的拟南芥不同，只有GV3101对法氏囊-巴斯德菌有较高的转化率。此外，我们还考察了不同浓度的蔗糖和表面活性剂Silwt L-77对法氏囊酵母转基因植株产生效率的影响，确定了一种含有10%(w/v)蔗糖和0.02-0.05%(v/v)Silwt L-77的高效培养基组分。通过Southern杂交，验证了标记基因已整合到植物基因组中，且导入基因在下一代遗传稳定。(C)2007 Elsevier B.V.保留所有权利。
Capsella bursa-pastoris is an attractive model system for evolutionary and developmental biology. To facilitate future studies on gene function, the 'floral dip' method was adapted to achieve germline transformation of C bursa-pastoris. The GFP and BASTA-resistance (BAR) genes were used as markers for screening or selecting, respectively, putative transgenic C bursa-pastoris plants and the beta-glucuronidase (GUS) gene as well as the GFP gene for monitoring transgene expression level. We tested two Agrobacterium strains, LBA4404 and GV3101, for their ability to transform C bursa-pastoris. In contrast to Arabidopsis thaliana, for which both strains were able to transform different ecotypes, only GV3101 gave satisfactory transformation rates with C bursa-pastoris. Furthermore, we evaluated the effects of different concentrations of sucrose and the surfactant Silwet L-77 on the efficiency to generate transgenic C bursa-pastoris plants and identified an efficient medium containing 10% (w/v) sucrose and 0.02-0.05% (v/v) Silwet L-77. Using Southern hybridisation, we confirmed the integration of the marker gene in the plant genome and the stable heredity of the introduced genes in the next generation. (C) 2007 Elsevier B.V. All rights reserved.