Kathiresan S, Chandrashekar A, Ravishankar G A, Sarada R
Plant Cell Biotechnology Department, Central Food Technological Research Institute, A Constituent Laboratory of the Council of Scientific and Industrial Research (CSIR), Mysore-570020, India.
J Phycol. 2009 Jun;45(3):642-9. doi: 10.1111/j.1529-8817.2009.00688.x. Epub 2009 Jun 1.
The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed β-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.
本文报道了首次成功利用携带编码GUS（β-葡萄糖醛酸酶）、GFP（绿色荧光蛋白）和hpt（潮霉素磷酸转移酶）基因的双元载体，通过农杆菌介导对绿藻雨生红球藻进行转化。在含有10 mg·L⁻¹潮霉素的培养基上获得的抗性菌落能够表达β-葡萄糖醛酸酶。当用荧光显微镜观察潮霉素抗性细胞时，可观察到GFP发出的黄绿色荧光。PCR成功地从转化细胞中扩增出hpt（407 bp）和GUS（515 bp）基因片段，而Southern杂交表明潮霉素基因已整合到雨生红球藻的基因组中。扫描电子显微镜显示，农杆菌感染后雨生红球藻的细胞壁发生了改变。此次通过农杆菌实现的转化无需乙酰丁香酮处理或细胞损伤。这种藻类的稳健转化方法将为调控与食品、制药和营养保健品行业相关的许多重要途径铺平道路。
