萝卜抗真菌蛋白1(Rs-AFP1)在大肠杆菌中的融合表达及其对大丽轮枝菌抑制活性的研究(英文)

利用聚合酶链式反应 (PCR)获得了萝卜 (RaphanussativusL .)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去除了N_端信号肽序列的片段分别装入原核表达载体pET_32b( )中 ,在大肠杆菌中表达 ,发现带有信号肽的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 2 7kD的Rs_AFP1的融合蛋白。用凝血酶处理融合蛋白以去除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 (VerticilliumdahliaeKleb .)的生长受到抑制 ,分别比加入对照细菌蛋白和PBS下降 5 7.5 %和 6 9.8% ;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。

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

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.