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

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。

In order to study the expression and the activity of inositol phosphorylceramide syntbase （BcAUR1 gene） in Botry＇tis cinerea, we amplified BcAUR1 by RT-PCR from Botrytis cinerea, using the special primers with FLAG and BamH Ⅰ/Xho Ⅰ restriction sites. Recombinant pYES2-BcAUR1 was constructed to transform into Saccharomvces cerevisae △yorl 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 Ayorl of Saccharomyces cerevisae. IPC synthase enzyme activity of pYES2-BcA UR1 transformants significantly increased and was approximately double than no-load BcA UR1 transformants. The low concentration of Aureobasidin A could inhibit growth of no-load BcAUR1 transformants, but pYES2-BcAUR1 induced by Aureobasidin A. transformants could resist fungal growth inhibition which was