Preparation and regenerating technology for protoplasm of rainy red ball alga

The invention relates to a microalgae, especially Haematococcus pluvialis protoplast preparing and regenerating technique. It uses a preprocessing agent prepared by acidic buffer solution, EDTA and dithiothreitol (DTT) to process microalgae cells at 25-34 deg.C for 30-60 min; uses a compound high-permeability enzyme composed of NaCl, KCl, MgSO4, CaCl2, cellulose, snailase, pectase and acidic buffer solution to eliminate cell walls so as to make the microalgae protoplast; using a storage solution composed of BBM culture solution, glucose and sucrose to store the protoplast, diluting and separating on to a regenerative culture medium containing 0.1mol/L glucose, 0.1mol/L sucrose and 0.1-0.5ppm melissyl alcohol for regenerative culture; keeping relative humidity greater than 60% and the temperature at 25 deg.C, and keeping illumination. The prepared protoplast is transparent and has high activity and birth colors and relatively circular in shape, the regenerative cycle can be shortenedby 1/3, the regeneration ratio is increased by 70%, and it can used for protoplast fusion, gene conversion, etc. 本发明涉及一种微藻，特别是雨生红球藻原生质体制备和再生的技术。该技术通过用酸性缓冲液、EDTA和二硫苏糖醇配制而成的预处理剂，25～34℃恒温处理微藻细胞30～60min；用NaCl，KCl，MgSO#-[4]，CaCl#-[2]，纤维素酶，蜗牛酶，果胶酶和酸性缓冲液组成的复合高渗酶溶液去除细胞壁制成微藻原生质体；用BBM培养液、葡萄糖和蔗糖组成的保存液保存原生质体，稀释后分离到含有0.1mol/L葡萄糖、0.1mol/L蔗糖、0.1～0.5ppm三十烷醇再生培养基上进行再生培养；保持相对湿度大于60％，温度为25℃，持续光照。本发明操作简便，设备要求低，原生质体制备率超过80％，制备的原生质体透明、活力高，具有鲜艳的颜色，形态较圆，再生周期可缩短三分之一，再生率提高70％，可供于原生质体融合和基因转化等操作。