根据Gen Bank数据库报道的林烟草(Nicotiana sylvestris)PGIP基因c DNA序列,设计特异性引物,从普通烟草(Nicotiana tabacum)种植品种小黄金中分离得到了其g DNA序列,分析结果发现烟草PGIP基因没有内含子序列。信号肽分析结果表明,烟草PGIP基因含有一段长28个氨基酸的信号肽。将去除信号肽的烟草PGIP基因片段亚克隆至枯草芽孢杆菌表达载体p HT43中,转化枯草芽孢杆菌菌株WB600并进行IPTG诱导。SDS-PAGE电泳表明,重组菌株可成功表达目的蛋白质,分子质量符合预期大小;琼脂扩散试验结果发现,表达的烟草PGIP蛋白质能够明显抑制辣椒疫霉PGs的活性。
The Nicotiana tabacum PGIP gDNA from the cultivar "xiaohuangjin" was cloned basing on one PGIP gene from the Nicotiana sylvestris. Sequence analysis showed that the gDNA of tobacco PGIP was identied to the eDNA sequence, with no introns. The tobacco PGIP gene was submitted to a online program for searching the signal peptide sequence. The results showed that the tobacco PGIP gene had a 28 amino acids signal sequence. Subsequently, the mature peptide sequence was subcloned into pHT43 expression vector and transferred into Bacillus subtilis WB600. After induced using IPTG, SDS-PAGE analysis showed that the recombinant protein expressed successfully with the expected molecular weight. Then the activity of the recombinant protein was determined by using AGAR double diffusion experiments. The results indicated that the recombi- nant PGIP of tobacco could inhibit the activity of PGs from Phytophthora capsici obviously.