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以从正在萌发的诸葛菜种子中提取总RNA反转录成的cDNA作为模板,进行PCR扩增,获得了160 bp的植物防御素cDNA片段。然后将该片段克隆到pMD18-T栽体。经测序后将片段酶切回收,再克隆到GTK原核表达载体中。经0.1 mmol/L IPTG诱导表达,获得了与理论值相符的32 kD的融合蛋白质条带。用GST单克隆抗体做第一抗体进行Western-Blot检测,获得阳性结果。这为诸葛菜植物防御素基因功能的研究提供了基础。
In this study, 160 bp cDNA fragments of plant defensins were obtained by PCR amplification of total RNA reverse transcribed from the germinating seeds of Brassica juncea. This fragment was then cloned into pMD18-T vector. After sequencing, the fragment was digested and recovered, and then cloned into the prokaryotic expression vector GTK. After induction with 0.1 mmol / L IPTG, a band of 32 kD fusion protein was obtained, which was consistent with the theoretical value. Western blotting with GST monoclonal antibody as the primary antibody yielded positive results. This provides a basis for the study of the function of plant defensin genes.