产琥珀酸放线杆菌的原生质体制备与再生

Protoplast Preparation and Regeneration of Actinobacillus succinogenes

基因组重组技术是一项重要的菌种改造技术,原生质体制备和再生是进行基因组重组的前提和基础。目前少有关于产琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC2650原生质体研究的报道。为了优化该菌的原生质体制备和再生条件,及利用基因组重组技术构建优良菌种提供参考,研究了甘氨酸预处理,菌龄,酶浓度,作用时间,温度对产琥珀酸放线杆菌原生质体制备和再生的影响,并考察了不同渗透压稳定剂对其再生的影响。结果表明,菌体在添加了0.6mg/ml甘氨酸的TSB培养基中培养5h后收集,用SMM稀释到OD660=1.0,用0.025mg/ml溶菌酶在37℃下酶解45min制备原生质体,将原生质体涂布于含0.3mol/L蔗糖的再生培养基中,再生率最大,达到40.9%。确定了产琥珀酸放线杆菌原生质体制备和再生的最佳条件,所用的原生质体制备的方法对琥珀酸的产生没有影响,这为进一步开展该菌的原生质体诱变及基因组重组等研究奠定了基础。

Genome shuffling was an important technology of strain improvement.The effects of glycin concentration,cell age,lysozyme concentration,the operational time and temperature were conducted to optimize the conditions of protoplast formation and regeneration of Actinobacillus succinogenes CGMCC 2650.The effects of the different osmotic stabilizing agents on regeneration were also examed.The protoplast yield was the highest under these conditions：The bacteria was cultivated in Tryptic Soy Broth（TSB）medium containing 0.6 mg/ml glycine for 5 h,then collected and diluted with SMM until the optical density（660nm）reached 1.0.The diluent was treated by 0.025mg/ml lysozyme at 37℃ for 45 min and plated on the TSB medium with 0.3 mol/L sucrose as osmotic stabilizer.The regeneration rate was up to 40.9%.This provides the optimized conditions of protoplast preparation and regeneration for genome shuffling of A.succinogenes.