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J. Hefei Univ. Tech. (Nat. Sci.)

合肥工业大学学报(自然科学版)

JOURNAL OF HEFEI UNIVERSITY OF TECHNOLOGY (NATURAL SCIENCE)

Competent Authorities: Ministry of Education of the People's Republic of China

Sponsor by: Hefei University of Technology

ISSN 1000-1646, CN 34-1001/N

Prokaryotic expression of BcMid1 and its interaction with chlorogenic acid

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BcMid1 蛋白原核表达及其与绿原酸的作用

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Jul.2023, Vol.46 No.7: 986-990

DOI: 10.3969/j.issn.1003-5060.2023.07.019

Authors

MA Zhitao, HU Tingting, ZHANG Danfeng, YE Yingwang

(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

Abstract and Keywords

Abstract: Mid1 protein is a fungal calcium channel protein that plays an important role in maintaining cellular calcium(Ca $ ^{2+} $) homeostasis. To study the relationship between the inhibitory effect of chlorogenic acid on the growth of Botrytis cinerea and Ca $ ^{2+} $, a plate inhibition experiment was applied to observing the effects of exogenous Ca $ ^{2+} $ on B. cinerea growth with the presence of chlorogenic acid. A BcMid1 prokaryotic expression vector was constructed and the BcMid1 protein was expressed in E. coli Rosetta. The fluorescence characteristics of chlorogenic acid were used to investigate the in vitro binding of BcMid1 protein to chlorogenic acid. The results showed that Ca $ ^{2+} $ could reduce the inhibition of chlorogenic acid on the growth of B. cinerea. The pET-28a::BcMid1 recombinant vector was successfully constructed and BcMid1 protein was expressed in Rosetta strain. SDS-PAGE and fluorescence quantification showed that chlorogenic acid and BcMid1 protein could bind, and their binding affinity was $ 3.93 \times 10^{4} $ mol/L. These results indicate that the inhibition of chlorogenic acid on B. cinerea is associated with Ca $ ^{2+} $ influx of B. cinerea cells, and BcMid1 protein is a potential target of chlorogenic acid. It lays a foundation for further study of the antifungal mechanism of chlorogenic acid on B. cinerea.

Keywords: Botrytis cinerea; Mid1 gene; chlorogenic acid; prokaryotic expression; fluorescence

作者信息

马志桃，胡婷婷，张丹凤，叶应旺

（合肥工业大学食品与生物工程学院，安徽合肥230601）

摘要和关键词

摘要: Mid1 蛋白是真菌钙通道蛋白, 在维持细胞钙稳态方面发挥着重要作用。文章为探讨绿原酸对灰霉 (Botrytis cinerea) 生长的抑制与钙之间的关系, 利用平板实验观察绿原酸存在条件下外源钙离子对灰霉生长的影响, 并构建 BcMid1 原核表达载体, 在大肠杆菌 Rosetta 菌株中诱导 BcMid1 蛋白表达, 利用绿原酸的荧光特性探究 BcMid1 蛋白与绿原酸的体外结合情况。结果显示, 钙离子可以降低绿原酸对灰霉的生长抑制。成功构建的 pET-28a::BcMid1 重组载体可以在 Rosetta 菌株中表达 BcMid1 蛋白, SDS-PAGE 和荧光定量检测发现绿原酸和 BcMid1 蛋白可以结合, 结合常数为 $ 3.93 \times 10^{4} $ mol/L。结果表明, 绿原酸对灰霉的抑制与钙摄入相关, 并且 BcMid1 蛋白是绿原酸的潜在作用靶点。该文为进一步研究绿原酸对灰霉的抑制机理奠定了基础。

关键词: 灰霉；Mid1基因；绿原酸；原核表达；荧光

Funding

国家自然科学基金资助项目（31500214）

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