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目的从川西獐牙菜Swertia mussotii中克隆牻牛儿基焦磷酸合成酶(Sm GPPS)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Sm GPPS基因序列,设计特异性引物,通过RT-PCR扩增得到c DNA序列,通过生物信息对该序列进行分析;构建原核表达载体p ET-28a-Sm GPPS,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Sm GPPS基因在川西獐牙菜不同组织中的表达强度。结果 Sm GPPS c DNA全长1 119bp,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。Sm GPPS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Sm GPPS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。
Objective To clone the coding gene of geranyl pyrophosphate synthetase (Sm GPPS) from Swertia mussotii, and to analyze its bioinformatics and expression of this gene. Methods According to Sm GPPS gene sequence of Swertia chinensis transcriptome, specific primers were designed and amplified by RT-PCR. The sequence of c DNA was analyzed by bioinformatics. The prokaryotic expression vector p ET-28a-Sm GPPS was constructed, Transformed into E. coli BL-21 (DE3) and expressed under the induction of 1 mmol / L IPTG at 37 ° C. Semi-quantitative RT-PCR method was used to detect the expression intensity of Sm GPPS gene in different tissues of Swertia mandshurica. Results The Sm GPPS c DNA was 1 119 bp in length and encoded 372 amino acids. The protein secondary and tertiary structures were analyzed and predicted. Sm GPPS proteins are highly similar to GPPS proteins in other plants. SDS-PAGE results showed that the expressed protein and expected protein size. Semi-quantitative RT-PCR results showed that Sm GPPS expression in leaves the highest. Conclusion The results provide the basis for further study of the function of this gene and the improvement of the yield of iridoids in Swertia chuanchia by genetic engineering.