Pratheesh P T, Vineetha M, Kurup G Muraleedhara
School of Biosciences, Mahatma Gandhi University, Kottayam, 686560, Kerala, India,
Mol Biotechnol. 2014 Jun;56(6):507-15. doi: 10.1007/s12033-013-9720-2.
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 μM 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.
近年来，基于藻类的重组蛋白生产引起了极大的关注。由于缺乏能够进行异源基因整合和表达的高效且经济高效的转化技术，藻类表达系统的发展早期受到了阻碍。农杆菌介导的遗传转化方法的最新进展有望成为解决这些问题的理想方案。我们已经开发出一种用于莱茵衣藻微藻的农杆菌介导遗传转化的高效方案。在含有100μM乙酰丁香酮和1mM甘氨酸甜菜碱的TAP诱导培养基（pH 5.2）中对农杆菌进行预处理，并用诱导后的农杆菌感染衣藻，极大地提高了转化频率。与之前的报道相比，该方案发现选择培养基上的转基因事件数量增加了一倍。PCR成功用于从转化细胞中扩增hpt和GUS基因的片段，而Southern印迹证实了GUS基因整合到莱茵衣藻的基因组中。RT-PCR、Northern印迹和GUS组织化学分析证实了GUS基因在衣藻转基因细胞系中的表达。该方案为微藻提供了一种快速、高效、经济且高频的转化方法。
