草酸青霉可重复利用筛选标记5'氟乳清酸的构建

Marker Recycling Via 5-fluoroorotic Acid Counter-selection in Penicillium oxalicum

草酸青霉是纤维素酶高产真菌,也是科学研究的重要真菌之一。借助基因工程技术手段对工业真菌进行分子改造,可以有效提高菌株在工业生产中的经济效益,对工业菌株进行基因改造需要大量的筛选标记,目前草酸青霉中可用的筛选标记较少,因此需要构建一个草酸青霉可重复利用筛选标记转化系统。本研究构建以乳清酸核苷-5’-磷酸脱羧酶基因(pyrG)作为选择标记,以草酸青霉pyrG缺失菌株为宿主菌构建转化系统。首先,构建了含有草酸青霉pyrG表达框及其终止子TT1重复序列的可重复利用pyrG筛选标记。然后,以pyrG筛选标记作为选择标记,以草酸青霉pyrG缺失菌株△ku70△pyrG(pyrG::kan)为受体菌株,敲除△ku70△pyrG(pyrG::kan)菌株中的kan基因,获得菌株△ku70△kan(kan::R-pyrG)。最后,利用筛选标记中的pyrG终止子重复序列发生自我剪切,利用氟乳清酸(5-FOA)的筛选,获得pyrG缺失,kan抗性基因缺失菌株△ku70△R-pyrG。本研究成功建立以pyrG为筛选标记,草酸青霉pyrG缺失菌株为受体菌的可重复利用筛选系统。

Penicillium oxalicum is one of important fungi for scientific research due to its high-yielding of cellulase.A great deal selection markers were required when this fungal was engineered to explore its economic benefits for industrial production with the help of genetic engineering modification.Therefore,it is necessary to build a reusable selection marker transformation system.In this study,a lactate nucleoside-5’-phosphate decarboxylase gene(pyr G)was used as a selection marker,and a transformation system was constructed in P.oxalicum.First,a reusable pyr G screening marker was constructed containing the P.oxalicum pyr G expression cassette and its terminator TT1 repeat.Then,byusing the pyr G selection marker as a selection marker,the kan gene of the△ku70△pyr G(pyr G::kan)strain was knocked out in△ku70△pyr G(pyr G::kan),and the strain△ku70△kan(kan::R-pyr G).Finally,self-shearing was performed by using the pyr G terminator repeat sequence in the selection marker,and the pyr G deletion,kan resistance gene deletion strain△ku70△R-pyr G was obtained by screening with fluoroorotic acid(5-FOA).A reusable screening system using pyr G as a screening marker and a P.oxalicum pyr G deletion strain as a recipient strain was successfully established.