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

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。

Abstract:

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.