Position: Home > Articles > Overexpression of Ace-AMP1 in Pichia pastoris on enhancing the inhibition of blue mold on pears
Journal of Zhejiang University (Agriculture and Life Sciences)
2019
(1)
39-46
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
作 者：
林明;姜路花;余挺
单 位：
淳安县农业技术推广中心;浙江大学生物系统工程与食品科学学院
关键词：
Ace-AMP1抗菌肽;毕赤酵母;青霉病;采后生物防治
摘 要：
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。
译 名：
Overexpression of Ace-AMP1 in Pichia pastoris on enhancing the inhibition of blue mold on pears
作 者：
LIN Ming;JIANG Luhua;YU Ting;College of Biosystems Engineering and Food Science, Zhejiang University;Chun'an County Agricultural Technology Extension Center;
单 位：
LIN Ming%JIANG Luhua%YU Ting%College of Biosystems Engineering and Food Science, Zhejiang University%Chun'an County Agricultural Technology Extension Center
关键词：
Ace-AMP1 antimicrobial peptide;;Pichia pastoris;;Penicillium expansum;;postharvest biocontrol
摘 要：
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 pPICZα A vector,which was designated as pPICZ α A/Ace-AMP1. The recombinant vector was then transformed P. pastoris through electroporation. The results of real-time quantitative polymerase chain reaction(RT-qPCR) and Westernblotting 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(109 μg/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.
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