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产黄青霉(Penicillium chrysogenum)是?-内酰胺类抗生素的主要工业生产菌,高效地对其基因进行改造有利于深入研究产黄青霉的代谢机制,进而提高效价、产率和得率。高产产黄青霉基因测序的完成为菌株定向改造提供了分子基础,但是要实现快速的定向菌株改造还需要高效的基因操作系统。本试验采用土壤农杆菌(Agrobacterium)转化法高效构建了海藻糖循环途径tps1、tps2敲除菌株P.chrysogenum-Δtps1和P.chrysogenum-Δtps2。土壤农杆菌为高毒力的根癌农杆菌AGL-1,载体为p Green II-0179,筛选标记为博来霉素,转化对象为产黄青霉孢子。在本试验条件下农杆菌转化法的转化效率可达60%,其中发生同源重组的效率为25%。本研究为后续探究海藻糖循环在高产产黄青霉中的生理学意义提供了有益的参考。
Penicillium chrysogenum is a major industrial producer of? -lactam antibiotics, and its efficient gene modification will help to further study the metabolic mechanism of Penicillium chrysogenum, thus improving the potency, yield and yield . The completion of high-yielding P. chrysogenum gene sequencing provided the molecular basis for directional transformation of the strain, but an efficient gene manipulation system is also needed to achieve rapid targeted strain transformation. In this experiment, trehalose cycle pathway tps1 and tps2 knockout strains P. chrysogenum-Δtps1 and P. chrysogenum-Δtps2 were efficiently constructed by Agrobacterium transformation. Agrobacterium tumefaciens is Agrobacterium tumefaciens AGL-1 with high virulence, vector p Green II-0179, selection marker bleomycin, and transformation target P. chrysogenum spores. Under the experimental conditions, the transformation efficiency of Agrobacterium transformation method can reach 60%, wherein the efficiency of homologous recombination occurs is 25%. This study provides a useful reference for further study on the physiological significance of trehalose cycle in high-yield P. chrysogenum.