An Efficient Protocol for the Agrobacterium-mediated Genetic Transformation of Microalga Chlamydomonas reinhardtii
An Efficient Protocol for the Agrobacterium-mediated Genetic Transformation of Microalga Chlamydomonas reinhardtii
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DOI：
10.1007/s12033-013-9720-2
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发表时间：
2014-06-01
影响因子：
2.6
通讯作者：
Kurup, G. Muraleedhara
中科院分区：
文献类型：
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作者：
Pratheesh, P. T.;Vineetha, M.;Kurup, G. Muraleedhara
近年来，以藻类为基础的重组蛋白生产获得了极大的兴趣。由于缺乏高效、经济的外源基因整合和表达转化技术，藻类表达系统的发展一直受到阻碍。近年来发展起来的农杆菌介导的遗传转化方法有望成为解决这些问题的理想方法。我们开发了一种高效的农杆菌介导的微藻莱茵衣藻遗传转化方案。在含有100 μ M乙酰紫丁香酮和1 mM甜菜碱的TAP诱导培养基（pH 5.2）中预处理农杆菌，并用诱导的农杆菌感染衣藻，可显著提高转化频率。与以前的报道相比，该方案使选择介质上的转基因事件数量增加了一倍。PCR成功地扩增了转化细胞的hpt和GUS基因片段，而Southern blot证实GUS基因已整合到莱茵哈特氏菌的基因组中。RT-PCR、Northern blot和GUS组织化学分析证实了转基因衣藻细胞系中GUS基因的表达。该协议为微藻提供了一种快速、高效、经济、高频的转化方法。
Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 mu M acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.