提高根癌农杆菌介导黑曲霉转化效率的研究

Improvement of Transformation Efficiency of Aspergillus niger Mediated by Agrobacterium tumefaciens

为提高根癌农杆菌(Agrobacterium tumefaciens)介导黑曲霉(Aspergillus niger)转化(ATMT)效率,本文对影响转化效率的分生孢子与农杆菌的比例、转化媒介、诱导溶氧、诱导时间、诱导时乙酰丁香酮(AS)浓度这5个因素进行单因素实验,最终确定提高其转化效率的最优条件为:根癌农杆菌诱导过程在400,μmol/L乙酰丁香酮终浓度条件下,100,m L三角瓶中28,℃、100,r/min振荡培养5,h,分生孢子与根癌农杆菌的比例为1﹕100,在硝酸纤维膜媒介上24,℃共培养48,h.在此优化条件下,黑曲霉的转化效率达到35个/107分生孢子.相比于优化前,转化效率提升了78%,,并且整合至黑曲霉基因组的外源潮霉素B抗性基因在转接15代以后仍能稳定遗传.

To improve the Agrobacterium tumefaciens-mediated transformation（ATMT）efficiency of Aspergillus niger,weperformed some single-factor tests for five factors：ratio of conidia to bacterium,transformation media,inducing dissolvedoxygen,inducing time and inducing concentration of acetosyringone（AS）.The optimum conditions to improve the transformationefficiency were as follows：A tumefaciens was cultivated in 400,μmol/L AS at 28,℃ and 100,r/min for 5,h in100,mL conical flask;co-cultivation was conducted at a 1﹕100,ratio of conidia to bacterium on nitrocellulose membrane at24,℃ for 48,h.Under the above optimized conditions,the transformation efficiency of A.niger achieved 35,transformants/107,conidia. The transformation efficiency was increased by 78%, compared with that before optimization,and all the selectedhygromycin B resistant transformants were genetically stable after 15 subcultures on complete medium.