黑曲霉pyrG遗传转化系统的构建及应用

Construction and application of Aspergillus niger pyrG genetic transformation system

用于黑曲霉遗传操作的筛选标记基因非常有限,而基因改造往往涉及诸多基因的敲除或表达,有限的筛选标记基因难以满足需要。该文构建了以pyrG为筛选标记的黑曲霉遗传转化系统,并利用该系统使红色荧光蛋白rfp在黑曲霉中成功表达。首先,运用同源重组原理完全敲除pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,获得1株稳定遗传的尿苷/尿嘧啶营养缺陷型菌株黑曲霉ng-1。然后构建了以pyrG作为回补标记,以三磷酸甘油醛脱氢酶启动子gpdA来调节rfp基因表达的质粒,将质粒转入农杆菌AGL1并侵染黑曲霉菌株ng-1,成功获得了黑曲霉rfp表达菌株,表明pyrG筛选标记可成功用于黑曲霉的遗传转化。

Few selective marker genes are used in the genetic operation of Aspergillus niger. However, genetic modification often involves the knockout or expression of many genes, and the limited selective marker genes are difficult to meet the needs. In this study, genetic transformation system was constructed using pyrG as selective marker in A. niger and the red fluorescent protein rfp in A. niger was expressed using this system. First, the pyrG gene was knocked out by targeted gene replacement using Agrobacterium tumefaciens mediated transformation. The stable genetic uridine/uracil auxotrophic strain A. niger ng-1 was obtained on medium containing 5-fluoroorotic acid and uridine/uracil. Then we constructed the rfp gene-expression plasmid which contained the rfp gene driven by the glyceraldehyde triphosphate dehydrogenase promoter gpdA and the selective marker pyrG. The plasmid was transferred to A. tumefaciens AGL1 and infected the A. niger ng-1, so as to obtain rfp gene-expression A. niger strains. This work showed that the genetic transformation system using the pyrG as a selective marker is effective and feasible in A. niger.