响应面法优化紫球藻原生质体的制备

Optimization of protoplast production fromPorphyridium cruentum with response surface method

[目的]优化紫球藻细胞原生质体制备条件。[方法]利用Box-Benhnken的中心组合实验设计,进行三因素三水平的实验设计。[结果]得到二次回归方程:Y=55.10+5.19X1-0.075X_2+1.76X_3-0.85X_1X_2-3.13X_1X_3-4.20X_2X_3-9.09X_1~2-6.66X_2~2-7.49X_3~2,确定了混合酶浓度(X1)、纤维素酶和果胶酶的比例(X2)和pH(X3)三因素对紫球藻原生质体制备率(Y)的组合效应,得到了混合酶浓度2.2%、纤维素酶与果胶酶体积比2∶3、pH 7、渗透压稳定剂为0.2mol·L-1 KCl、30℃下轻微振荡、酶解6h的最优制备条件,在此条件下得到68.5%的原生质体制备率。[结论]本研究提高了紫球藻原生质体的制备率,能够满足后续基因工程和遗传育种研究的需要。

[Objective]Present study was conducted to determine an optimal condition for protoplast preparation from Porphyridium cruentum with response surface methodology. [Methods] A model of Box Behnken central composite design was utilized with three factors at three levels to detect the greatest protoplast formation rate within the experi mental range. [Results] An equation was obtained as follows to fit the empirical evidence between protoplast formation rate （Y） and concentration of Cellulose and Macerozyme （X1）, Cellulose / Macerozyme （X2）, and pH （X3）. Y一55.10＋5.19X10.075Xz＋1.76X30.85X1Xz一3.13x1X3—4.20X2X3—9.09X1,2—6.66X2 2一7.49X3 2. When algae were cultured in 0.2 mol-L 1 KC1 buffer with pH 7 and were gently shaken for 6h at 30~C , , the optimal formulation was obtained with concentration of Cellulose and Macerozyme at 2.2~ and Cellulose / Macerozyme, at 2： 3. The high est protoplast formation rate of 68.5 c^00 was reached with these optimal parameters. EConclusion] Study results demon strated that the protoplast formation rate of Porphyridium cruentum was considerably improved under mentioned opti real condition, which could benefit the research of bioengineering and genetic breeding.