小立碗藓原生质体细胞壁再生过程观察

Protoplast cell-wall regeneration in Physcomitrella patens

【目的】小立碗藓具有遗传背景清楚、活体观察方便、生活史短、再生能力强等特点,是研究发育的理想模式植物。【方法】应用0.5%的崩溃酶将生长7 d的原丝体酶解,得到新鲜原生质体,先过滤再用8%的甘露醇洗涤,在含有为8%的甘露醇的BCDA液体培养基内培养2 d和4 d。采用新鲜原生质体、再生2 d和再生4d的原生质体作材料,使用透射电镜,观察不同时期原生质体的细胞壁再生过程,并运用实时荧光定量PCR技术分析了细胞壁再生相关基因的表达。【结果】研究结果表明,原生质体培养2 d后开始有微纤丝发生,在原生质体培养4 d时形成细胞壁结构,细胞开始进行分裂。分析细胞壁再生相关基因的表达量,发现在新鲜原生质体中基因表达量最低,原生质体培养2 d和培养4 d后,基因表达量均比在新鲜原生质体中的表达量高。【结论】小立碗藓原生质体在液体培养基中培养2 d微纤丝发生;原生质体培养4 d形成完整的细胞壁结构。

【Objective】Physcomitrella patens,with known genetic background,convenient in vivo observation,short life cycle and strong regeneration ability,is an ideal model plant to study developmental biology.【Methods】To produce protoplasts,seven days old protonemata were digested with 0. 5% driselase. After filtration and washing,the protoplasts were cultured on liquid BCDA medium with 8% mannitol for 2 days and 4 days. The cell-wall regeneration was observed using transmission electron microscope,and the gene expression was analyzed by Quantitative Real-time PCR.【Results】The primary-wall microfibrils were formed in the tissues of protoplast regeneration for 2 days. The cell walls were formed and cells division occurred during protoplast regeneration for 4 days. Furthermore,we analyzed the m RNA expression of 6 genes which are involved in cell-wall regeneration. Quantitative PCR showed that the expression of the 6 genes was up-regulated in the tissues of protoplast regenerated for 2 days and 4 days.【Conclusion】The primary-wall microfibrils were formed in the process of protoplast regenerated for 2 days in the liquid medium,and the intact cell-wall structure were formed during protoplast regenerated for 4 days in P. patens.