灰葡萄孢菌AUR1基因真核表达载体的构建、表达及酶活性分析
Construction, expression and enzymatic activity analysis of AUR1 eukaryotic expression vector of Botrytis cinerea
投稿时间:2012-07-31  
DOI:  
中文关键词:pYES2穿梭载体,灰葡萄孢菌,肌糖磷脂酰神经酰胺合成酶,短梗霉素A,酶活力
英文关键词:pYES2 shuttle plasmid, Botrytis cinerea, inositol phosphorylceramide (IPC) synthase, Aureobasidin A , enzyme activity
基金项目:国家自然科学基金 (No. 30760131) 资助。
作者单位
邱永春 新疆大学生命科学与技术学院新疆 乌鲁木齐 830046 
刘小平 新疆大学生命科学与技术学院新疆 乌鲁木齐 830046 
苟萍 新疆大学生命科学与技术学院新疆 乌鲁木齐 830046 
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中文摘要:
      为了研究灰葡萄孢菌肌糖磷脂酰神经酰胺合成酶 (BcAUR1基因) 的表达及酶活性,采用RT-PCR方法,利用含有FLAG标签以及BamHⅠ、XhoⅠ酶切位点的AUR1特异引物从灰葡萄孢菌中扩增得到BcAUR1基因。将BcAUR1基因与穿梭质粒pYES2重组,得到pYES2-BcAUR1质粒采用醋酸锂转化法导入酿酒酵母尿嘧啶突变菌株Δyor1中,Western blotting检测肌糖磷脂酰神经酰胺 (IPC) 合成酶表达,HPLC检测IPC合成酶活力。结果显示pYES2-BcAUR1在酿酒酵母尿嘧啶突变菌株Δyor1中获得表达,pYES2-BcAUR1转化子IPC合成酶活性显著增高,比空载转化子约提高1倍。低浓度的AbA能够抑制空载pYES2酵母转化子生长,但pYES2-BcAUR1酵母转化子能抵抗AbA对菌体生长的抑制。
英文摘要:
      In order to study the expression and the activity of inositol phosphorylceramide synthase (BcAUR1 gene) in Botrytis cinerea, we amplified BcAUR1 by RT-PCR from Botrytis cinerea, using the special primers with FLAG and BamHⅠ/XhoⅠrestriction sites. Recombinant pYES2-BcAUR1 was constructed to transform into Saccharomyces cerevisae Δyor1 by LiAC. The expression of inositol phosphorylceramide (IPC) synthase and its activity were detected by Western blotting and HPLC, respectively. The results show that pYES2-BcAUR1 could express in uracil?mutant Δyor1 of Saccharomyces cerevisae. IPC synthase enzyme activity of pYES2-BcAUR1 transformants significantly increased and was approximately double than no-load BcAUR1 transformants. The low concentration of Aureobasidin A could inhibit growth of no-load BcAUR1 transformants, but pYES2-BcAUR1 transformants could resist fungal growth inhibition which was induced by Aureobasidin A.
邱永春,刘小平,苟萍.灰葡萄孢菌AUR1基因真核表达载体的构建、表达及酶活性分析[J].生物工程学报,2013,29(1):78~86
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