基于亚细胞定位的大豆和鹰嘴豆原生质体分离体系的建立与优化

Establishment and optimization of systems for protoplasts isolation of soybean and chickpea that used in subcellular location

摘要:

以湘豆3号大豆和Kabuli型鹰嘴豆幼嫩叶片为材料，研究了酶解种类及配比、酶解液中甘露醇浓度、酶解液pH和酶解时间对两种豆科植物幼嫩叶片原生质体产量和存活率的影响。结果表明，湘豆3号大豆叶片在含有0.5%纤维素酶Onozuka R-10、0.8%半纤维素酶Hemicellulase、0.8%离析酶Macerozyme R-10、0.4%果胶酶Pectolyase Y-23、0.1% 2-(N-吗啡啉)乙磺酸 (MES) 和10%甘露醇的酶解液中 (pH 6.0)，27 ℃黑暗条件下恒温水浴振荡 (45 r/min) 酶解6 h，分离得到的原生质体产量和存活率最高；Kabuli型鹰嘴豆叶片在含有0.5%纤维素酶Onozuka R-10、0.8%半纤维素酶Hemicellulase、0.8%离析酶Macerozyme R-10、0.1% MES和10%甘露醇酶解液中 (pH 4.8)，27 ℃黑暗条件下恒温振荡水浴 (45 r/min) 7–8 h，分离得到的原生质体产量和存活率最高。通过上述件分离到的湘豆3号大豆和Kabuli型鹰嘴豆幼嫩叶片原生质体用于亚细胞定位的效果最好。

Abstract:

Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.