[关键词]
[摘要]
目的 从川西獐牙菜Swertia mussotii中克隆牻牛儿基焦磷酸合成酶(SmGPPS)编码基因,并进行生物信息分析及该基因的表达研究。方法 根据川西獐牙菜转录组SmGPPS基因序列,设计特异性引物,通过RT-PCR扩增得到cDNA序列,通过生物信息对该序列进行分析;构建原核表达载体pET-28a-SmGPPS,转入大肠杆菌BL-21(DE3)中,在37℃、1 mmol/L IPTG诱导下进行表达。采用半定量RT-PCR方法检测SmGPPS基因在川西獐牙菜不同组织中的表达强度。结果 SmGPPS cDNA全长1 119 bp,编码372个氨基酸。并对其蛋白二级、三级结构进行了分析和预测。SmGPPS蛋白与其他植物中GPPS蛋白具有高度的相似性。SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致。半定量RT-PCR结果表明SmGPPS在叶中表达量最高。结论 为进一步研究该基因的功能和利用基因工程手段提高川西獐牙菜中环烯醚萜类化合物产量提供了基础。
[Key word]
[Abstract]
Objective To clone the geranyl pyrophosphate synthase gene from Swertia mussotii (SmGPPS), analyze the bioinformation of SmGPPS, and perform the gene expression. Methods According to the SmGPPS gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector pET-28a-SmGPPS was constructed and transformed into Escherichia coli BL-21 (DE3) for expression under 37℃ and induced by 1 mmol/L IPTG. The relative expression of gene SmGPPS in the leaf, stem, and flower of S. mussotii was also studied. Results The results showed that SmGPPS cDNA complete sequences had a length of 1 119 bp encoding 372 amino acid residues. And the protein secondary and tertiary structures were analyzed and forecasted. The SmGPPS protein shared high identity with other GPPS proteins of plants. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Relative RT-PCR analysis indicated that SmGPPS showed the highest transcript abundance in the leaf. Conclusion This work will provide a foundation for further functional research of SmGPPS protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
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[基金项目]
国家自然基金资助项目(81303303);天津市高等学校科技发展基金计划项目(20130203)