蓝藻球形体的分离、培养及再生

ISOLATION,CULTURE AND REGENERATION OF SPHEROPLASTS OF BLUE-GREEN ALGAE

在高渗溶液中,用0.05%溶菌酶和2—5mmol·1^(-1)EDTA 处理蓝藻柱孢鱼腥藻、多变鱼腥藻和组囊藻细胞。5—8h 后,70—90%的细胞转为对渗透压敏感的球形体(Spheroplast),又称原生质球。研究了藻的不同培养条件对球形体形成率的影响。测定了 EDTA 处理藻纽胞后外膜脂多糖的释放量。在高渗溶液中,藻细胞和经酶处理获得的球形体的光合放氧活性明显下降,固氮种类的固氮活性失去。饲养层法、固体混合法和含有0.5mg·1^(-1)BA 的液体悬滴培养的柱孢鱼腥藻的球形体,9天后出现再生藻落;在固体混合法培养中获得了组囊藻球形体的再生藻落。在第4天的悬滴培养物中,可以看到球形体发生第一次细胞分裂。再生藻细胞和酶处理物中残留细胞的抗溶菌酶特性有差异。

Growing cells of Anabaena cylindrica,A.variabilis and Anacystis nidulans were treated with 0.05% lysozyme (w/v) and 2—5mmol·1^(-1) EDTA,at 35℃ in hypertonic solution.Within 5—8h,about 70—90% of the cells were converted to osmotically sensitive spheroplasts.The effect of culture methods on the formation rate of spheroplasts was studied.The amount of lipopolysaccharides(LPS) released from outer membrane of cells treated with EDTA only was measured.The photosynthetic activity of the spheroplasts and cells suspended in hypertonic solution decreased obviously.The cells suspending in hypertonic solution lost their nitrogen- fixing activity.The spheroplasts of A.cylindrica plated on the agar feed-layer,embedded in the agar medium (0.8—1.5%) or suspended in the culture drop containing 0.5mg·1^(-1) BA formed the regenerated algae colonies after 9 days.The spheroplasts of A.nidulans embed- ded in the soft agar (0.8%) formed the regenerated algae colonies.Cell division of spheropla- sts suspended in the drop culture could be found on the 4th day of culture.Lysozyme-resis- tance of regenerated algae colonies was compared with that of the cell colonies developed from the residual cells in the lysozyme-treated solution.Spheroplasts of two species were in- duced to fusion with PEG and high pH/Ca,but the attempt failed.