原生质体融合选育高产多糖的罗尔阿太菌菌株

李鸿梅; 赵慧; 魏明; 闵伟红; 张桂弘; 刘景圣

doi:10.7671/j.issn.1001-411X.2015.04.017

原生质体融合选育高产多糖的罗尔阿太菌菌株

Breeding of Athelia rolfsii strain with high polysaccharide yield by protoplast fusion

摘要

摘要:
目的选育罗尔阿太菌多糖高产、草酸低产的优良罗尔阿太菌菌株.
方法采用原生质体融合技术，以罗尔阿太菌菌株AY6657741为原始菌株，以紫外和亚硝基胍复合诱变的2株多糖高产的诱变株Ar-1和Ar-2为双亲菌株; 通过正交试验优化原生质体的制备条件.
结果和结论 5.56 mmol·L^-1的β-巯基乙醇处理菌悬液，0.4 mol·L^-1的KCl作为渗透压稳定剂，3.1 mg·mL^-1的复合酶液(纤维素酶φ为2%、蜗牛酶φ为2%、溶壁酶φ为4%)，pH 5.5，32 ℃酶解55 min，原生质体形成率93.534%，再生率18.921%.0.4 g·mL^-1的聚乙二醇6000在30 ℃条件下融合25 min，选育出1株多糖高产、草酸低产的菌株，命名为AY-3.菌株AY-3多糖产量达24.673 g·L^-1，比原始菌株AY6657741的产糖量提高了54.42%，草酸质量浓度降低至0.281 g·L^-1，比原始菌株草酸的质量浓度降低了43.57%.

Abstract:
Objective To breed Athelia rolfsii strain with high yield of polysaccharide and low yield of oxalic acid.
Method The protoplast fusion technique was adopted with original strain AY6657741 and parental strains of high polysaccharide yield Ar-1 and Ar-2, which were screened from AY6657741 by NTG with UV.The orthogonal test was designed to obtain the optimal conditions for preparation of protoplast fu sion strains.
Result and conclusion The Ar-1 and Ar-2 were suspended by 5.56 mmol·L^-1 β-mercaptoethanol.Osmotic pressure stabilizer was 0.4 mol·L^-1 KCl.Ar-1 and Ar-2 were hydrolyzed by 3.1 mg·mL^-1 compound enzyme (2% cellulase, 2% snailase, 4% lywallzyme) for 55 min at pH 5.5, under the 32 ℃ condition.Protoplast formation rate and regeneration rate were 93.534% and 18.921% respectively.A strain, named AY-3, with high polysaccharide yield and low oxalic acid yield was screened by protoplast fusion under the condition of 0.4 g·mL^-1 polyethylene glycol 6000 for 25 min at 30 ℃. Compared with Athelia Rolfsii AY6657741, the polysaccharide yield of strain AY-3 increased by 54.42% (24.673 g·L^-1), and oxalic acid yield reduced by 43.57% (0.281 g·L^-1).

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