复合酶法高效制备紫菜原生质体的研究
Research of Efficient Preparation of Laver Protoplasts by Compound Enzymatic Method
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摘要: 以紫菜(Porphyra yezoensis)为研究对象,通过复合酶水解获得原生质体,研究酶解温度、加酶量、酶解时间、摇床转速、盐度、复合酶中组分比例、金属离子对酶解后原生质体数的影响。在单因素试验的基础上,以原生质体数为指标,选取因子与水平进行正交实验,对酶解条件进行优化研究。结果表明,最佳酶解条件为:酶解温度28 ℃、转速125 r/min、纤维素粗酶含量3%、琼胶酶12 mL、盐度4%、酶解时间100 min。同时发现一些金属离子对原生质体制备有抑制作用。在最优酶解条件下,得到的原生质体活率较高,即1 g紫菜样品经酶解得到的原生质体活率高达(16.88±0.22)×106个/g。综上,复合酶法制备紫菜原生质体反应条件温和、成本低廉,为紫菜的遗传育种以及在水产养殖中的应用研究提供理论依据。Abstract: Laver (Porphyra yezoensis) was chosen for enzymatic hydrolysis, and protoplasts were obtained by enzyme hydrolysis. Effects on the enzyme of temperature, the amount of enzyme, reaction time, rotary speed, salinity, the proportion of components in the enzyme and metal ion were studied by the number of laver protoplasts. Factors and levels were selected on the basis of single factor test, the optimization of hydrolysis conditions were obtained by orthogonal experiment with the number of protoplasts as the index. The results indicated that the optimal hydrolytic conditions were:Temperature 28℃, rotary speed 125 r/min, content of cellulose crude enzyme 3%, the agar enzyme 12 mL, salinity 4% and hydrolysis time 100 min. Protoplast formation inhibited by some metal ions were found simultaneously. Protoplasts obtained under the optimal hydrolysis conditions were high live rate, the number of protoplasts obtained from 1-g porphyra sample was (16.88±0.22)×106 cells/g.
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