库德毕赤酵母原生质体制备与再生  

Preparation and regeneration of pichia kudriavzevii protoplasts

作　　者：蔡爱华[1] 陈海珊[1] 周玉恒[1] 覃香香[1] 张厚瑞[1] 

机构地区：[1]中国科学院广西植物研究所广西植物功能物质研究与利用重点实验室,广西桂林541006

出　　处：《可再生能源》2012年第5期47-53,共7页Renewable Energy Resources

基　　金：国家自然科学基金项目(NO.20666002);广西自然科学基金项目(NO.2010GXNSFB013043;2010GXNSFA013060);广西植物研究所基本科研业务费资助项目(桂植业NO.09001)

摘　　要：库德毕赤酵母(Pichia kudriavzevii)对木质纤维素稀酸水解物3大代表性毒物——脂肪酸、呋喃及酚类化合物均有优良的降解活性,是纤维水解物耐毒发酵菌种改造重要的基因组资源。文章研究了库德毕赤酵母原生质体制备、再生的影响因素,确定了适宜的原生质体制备条件,旨在为通过基因组改组策略构建耐毒发酵菌株奠定基础。结果表明:选择对数生长中期的细胞,以0.8 mol/L甘露醇作为渗透压稳定剂,0.1%ED-TA-Na2+0.3%β-巯基乙醇预处理30 min,用3.0%蜗牛酶+0.5%纤维素酶在30℃下酶解2.5 h,其原生质体达到61%,原生质体再生率达到32%;毒物会强烈抑制原生质体再生,不宜直接以毒物作为再生前融合子的检出标记。Pichia kudriavzevii has a efficient degradation capability on fatty acid,furan,and phenols,which are the three main toxic byproducts of the lignocelluloses dilute-acid hydrolysate.n.The effect factors of protoplast preparation and regeneration of P.kudriavzevii was studied,and the optimized conditions was made sure.The results showed that when taking 0.8 mol/L mannitol as infltration stabilizer,the mid-log phase strains of P.kudriavzevii being pretreated with 0.1% EDTA-Na2＋0.3%Beta-mercaptoethanol for 30 min,following by an digestion in 3.0% snailase and 0.5% cellulase mixture system for 2.5 h at 30 ℃,the percentages of protoplast formation and regeneration of P.kudriavzevii could reach 61% and 32%,respectively.The regeneration could be strongly inhibited by toxic substance,which suggested that the latter should not be used as screening markers for the desired fusion directory.

关 键 词：库德毕赤酵母 木质纤维 稀酸水解物 毒物 原生质体 

分 类 号：TK6[动力工程及工程热物理—生物能] Q814[生物学—生物工程]