短短芽孢杆菌X23中edeB基因的克隆、原核表达及转录时相分析

Cloning, Prokaryotic Expression and Transcription Profile Analysis of edeB Gene in Brevibacillus brevis X23

短短芽孢杆菌(Brevibacillus brevis)X23可产生非核糖体肽类抗生素伊短菌素,已被广泛用于植物病害的生物防治。伊短菌素合成基因簇中,edeB基因参与调控伊短菌素的合成积累。本研究利用基因同源重组技术,以edeB基因作为外源基因的重组片段,构建了原核表达载体pET28a-edeB,转化至大肠杆菌BL21(DE)中进行诱导表达;通过转录组测序检测edeB基因在短短芽孢杆菌不同培养时间(12、18、24、30和36 h)的转录时相。结果表明:edeB基因全长为771 bp,编码256个氨基酸。聚丙烯酰胺凝胶电泳(SDS-PAGE)显示,在分子质量为30 kD处有一条特异条带,与生物信息学预测的EdeB蛋白的大小一致,且EdeB蛋白主要以包涵体的形式存在。转录时相分析结果表明,edeB基因在各个培养时间点均有表达,表达模式为先升高后降低,且在30 h时表达水平最高。这些结果为进一步研究edeB基因调控伊短菌素合成积累的分子机制奠定了基础,为短短芽孢杆菌高产伊短菌素菌株的构建提供了理论依据。

Brevibacillus brevis X23 can produce non-ribosomal antibiotic edeines, which have been widely used in biological control of plant diseases. The edeB gene in the edeine biosynthetic gene cluster regulates the synthesis of edeines. In this study, the prokaryotic expression vector pET28a-edeB was constructed by homologous recombination technology with edeB gene as the recombinant fragment of exogenous gene, and was transformed into Escherichia coli BL21(DE) for induced expression. The transcription profile of edeB gene in different culture times(12, 18, 24, 30 and 36 h) of B. brevis was detected by transcriptome sequencing. The results showed that the edeB gene was 771 bp in length, encoding 256 amino acids. SDS-PAGE electrophoresis showed that a specific band with a molecular weight of 30 kD appeared, which is in consistent with the bioinformatics prediction results. EdeB protein mainly existed in the form of inclusion bodies. The results of transcription profile showed that edeB gene was expressed at all culture times, and the gene expression showed a pattern of increasing at first and then decreasing with a peak at 30 h. These results laid a foundation for further study on the molecular mechanism of edeB gene regulating the synthesis of edeines, and provided a theoretical basis for the construction of high-yield edeine strains of B. brevis.