J Integr Plant Biol. ›› 2000, Vol. 42 ›› Issue (7): 703-707.

• Research Articles • Previous Articles     Next Articles

Fusion Expression of Raphanus sativus-Antifungal Protein 1 (Rs-AFP1) in Escherichia coli and Its Antifungal Activity on Verticillium dahliae

ZHOU Xiang-Jun, WANG Lun-Shan, LIN Zhi-Ping, JIA Jun-Wei, LU Shan, CHU Zhao-Qing, CHEN Xiao-Ya   


The Raphanus sativus L. antifungal protein 1 (Rs-AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET-32b(+), respectively. Subsequent expression showed that the Rs-AFP1 was produced in E. coli as a 27 kD fusion protein only when the N-terminal signal peptide was removed. After treatment with thrombin to remove part of the N-terminal His.tag sequence, the bacterially expressed Rs-AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil-born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs-AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.

周向军1  王仑山2  林芝萍1  贾军伟1 卢山1  储昭庆1 陈晓亚1*

(1. 中国科学院上海生命科学研究院植物生理研究所,植物分子遗传国家重点实验室,上海生命科学研究中心,上海200032;2.兰州大学生命科学学院,兰州730000)

摘要:利用聚合酶链式反应(PCR)获得了萝卜(Raqhanus sativus L.)抗真菌蛋白1(Rs-AFP1)基因编码区核苷酸序列。将整个阅读框架片段和云除了N-端信号肽序列的片段分别装入原核表达载体pET-32g(+)中,在大肠杆菌中表达,发现带有信号 的Rs-AFP1不能在大肠杆菌中表达,而当这一序列去除后,表达出约27kD的Rs-AFP1的融合蛋白。用凝血酶处理融合蛋白以云除N-端His.tag的部分序列,然后用处理后的融合蛋白进行了抑制真菌生长的实验。结果表明,在加入0.3g/L的Rs-AFP1的融合蛋白的培养液中,大丽轮枝菌(Vertivillium dahloae Kleb.)的生
长受到抑制,分别比加入对照细菌蛋白和PBS下降57.5% 和69.8%;孢子的萌发也受到抑制。显然,细菌表达的融合蛋白对大丽轮枝菌的生长有抑制作用。
关键词: 萝卜抗真菌蛋白1;真菌;大丽轮枝菌;原核表达

Key words: Raphanus sativus-antifungal protein 1 (Rs-AFP1), fungus, Verticillium dahliae, prokaryotic expression

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