农杆菌介导双孢蘑菇AS2796转化体系的优化

Optimization of Agrobacterium-mediated transformation conditions on strain of Agaricus bisporus AS2796

双孢蘑菇Agaricus bisporus是一种药食皆宜的食用真菌,具有极高的经济价值,近年来在双孢蘑菇全基因组测序完成后,功能基因组学的研究逐步提上日程。其中,高效的遗传转化体系作为技术基础成为研究重点。农杆菌介导的遗传转化体系作为一种高效的功能基因组研究方法,在双孢蘑菇中已经得到了广泛的应用,但是仍然存在着效率较低的问题有待解决。为了提高双孢蘑菇的农杆菌转化效率,找到双孢蘑菇遗传转化的最优条件,本实验首先对双孢蘑菇的潮霉素抗性压力进行了筛选,结果显示20mg/L的潮霉素浓度可以抑制菌丝生长,而50mg/L的潮霉素浓度则可以完全抑制菌丝生长。其次,对农杆菌菌株、菌液浓度、乙酰丁香酮浓度、菌丝培养时间以及浸染时间做了L16(45)的正交实验,通过对各因素影响的分析,得出对转化效率影响最大的因素为菌丝培养时间和农杆菌菌株,且当菌株为LBA4404、菌液浓度OD600=0.8、乙酰丁香酮浓度为100μmol/L、菌丝培养时间为40d、浸染时间为10min时,农杆菌介导的双孢蘑菇遗传转化效率最高。

Agaricus bisporus is an edible mushroom possessing very high economic value.Recent years,a high-efficiency functional genomic research method,Agrobacterium-mediated genetic transformation system,has been widely used for Agaricus bisporus,but the problems of low efficiency need to be improved.In this study,the method for improvement of transformation efficiency and finding the optimal genetic transformation conditions was proposed by detection of resistance of Agaricus bisporus to hygromycin.The results showed that hygromycin at concentration of 20mg/L could weaken the growth of mycelia,and at concentration of 50mg/L completely inhibit mycelia growth.The orthogonal test of L16(45)was carried out to analyse the effects of different strain of Agrobacterium,concentration of Agrobacterium,concentration of acetosyringone,culture duration of mycelia and infection duration of agrobacteria on optimizing the conditions of Agrobacterium-mediated genetic transformation in Agaricus bisporus.The results indicated that the culture duration of mycelia and selected strain of Agrobacterium were important factors influencing transformation efficiency.The highest genetic transformation efficiency was obtained by using Agrobacterium tumefaciens LBA4404 at concentration of OD600 0.8,under the acetosyringone concentration of 100mmol/L,mycelial culture duration of 40 days and infection duration of 10 minutes.