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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (1): 39-46    DOI: 10.3785/j.issn.1008-9209.2017.10.092
Plant protection     
Overexpression of Ace-AMP1 in Pichia pastoris on enhancing the inhibition of blue mold on pears
Ming LIN1(),Luhua JIANG2,Ting YU1,2()
1. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
2. Chun’an County Agricultural Technology Extension Center, Hangzhou 311700, China
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Abstract  

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 pPICZα 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 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 (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.

Key words： Ace-AMP1 antimicrobial peptide      Pichia pastoris      Penicillium expansum      postharvest biocontrol     
Received: 09 October 2017      Published: 28 March 2019
CLC:  Q 812  
  S 476  
Corresponding Authors: Ting YU     E-mail: 21513030@zju.edu.cn;yuting@zju.edu.cn
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Ming LIN
Luhua JIANG
Ting YU
Cite this article:

Ming LIN,Luhua JIANG,Ting YU. 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, 45(1): 39-46.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2017.10.092     OR     http://www.zjujournals.com/agr/Y2019/V45/I1/39


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

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pPICZα A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（109 μg/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。


关键词： Ace-AMP1抗菌肽,  毕赤酵母,  青霉病,  采后生物防治 
Fig. 1 Identification result of pPICZα A/Ace-AMP1 recom-binant plasmid by PCR
Fig. 2 Identification result of GS115/Ace-AMP1 recom-binant strain by PCR
Fig. 3 Protein content of GS115/Ace-AMP1 recombinant strain
Fig. 4 Relative expression amount of Ace-AMP1 for the recombinant strain GS115/Ace-AMP1
Fig. 5 Identification result of GS115/Ace-AMP1 recombinant strain by Western-blotting
Fig. 6 Control efficiency of recombinant strain GS115/Ace-AMP1 on Penicillium expansum in pear fruit
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