Ace-AMP1过表达毕赤酵母工程菌株的构建及其对梨青霉病的抑制作用

Overexpression of Ace-AMP1 in Pichia pastoris on enhancing the inhibition of blue mold on pears

为解决从洋葱种子中直接提取Ace-AMP1抗菌肽难度大、成本高、提取率低的问题,本研究根据毕赤酵母密码子的偏好性,优化合成了洋葱种子的Ace-AMP1抗菌肽基因,将其连接到表达载体pPICZαA中,并电转化毕赤酵母GS115感受态细胞进行表达。实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction, RT-qPCR)及蛋白质印迹法(Western-blotting)结果证实,Ace-AMP1过表达GS115构建成功,抗菌肽能在酵母体内稳定表达。对蛋白质表达水平的检测结果表明,GS115/Ace-AMP1经诱导后上清液中蛋白质质量浓度显著增加,并于84 h达到最大值(109μg/mL),而对照组则一直处于较低水平。对梨果实青霉病的抑制效果实验表明:GS115/Ace-AMP1能显著抑制扩展青霉(Penicillium expansum)在梨果实上的侵染;与对照组相比,青霉病发病率下降34%,平均病斑直径减小65%。本研究为Ace-AMP1抗菌肽的大规模生产及其抑制果实采后病害的开发利用奠定了基础。

Conventional methods of Ace-AMP1 antimicrobial peptide extraction from onion seeds are usually difficult,costly with a low extraction rate.To solve these problems,the original gene of antimicrobial peptide Ace-AMP1 was optimized according to the preference of Pichia pastoris and ligated into pPICZαA vector,which was designated as pPICZαA/Ace-AMP1.The recombinant vector was then transformed P.pastoris through electroporation.The results of real-time quantitative polymerase chain reaction(RT-qPCR)and Western-blotting confirmed that the Ace-AMP1 was successfully overexpressed in GS115,and the antibacterial peptide could be stably expressed in yeast cell.The protein expression level showed a significant increase in the supernatant after the induction,reached the maximum concentration(109μg/mL)at 84 h,while the control group still remained a low level.The biocontrol experiment indicated that the recombinant strain GS115/Ace-AMP1 could highly control the blue mold compared with the controls,which was 34%of disease incidence lower than the sterile distilled water treatment.The same trend was observed in the lesion diameter,which dropped by 65%with the GS115/Ace-AMP1 treatment compared with the control.Therefore,the GS115/Ace-AMP1 can be as an Ace-AMP1 peptide manufacturer,as well as a biocontrol agent.