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目的从黄芩愈伤组织中克隆查耳酮异构酶(CHI)基因,并对其进行生物信息学分析。方法从黄芩愈伤组织中提取RNA,反转录为cDNA,设计特异性引物,克隆得到黄芩查耳酮异构酶(sbCHI)基因。通过生物信息学对该基因蛋白的特征进行分析,使用MEGA5.1构建sbCHI与相关物种查耳酮异构酶基因的系统进化树。结果获得sbCHI基因(GeneBank登录号KP064512)全长648 bp,含有1个完整的开放阅读框,编码215个氨基酸。为不稳定亲水性蛋白,sbCHI编码蛋白相对分子质量为22 980,等电点(pI)为5.09,不含信号肽,无跨膜区,具有1个查耳酮超级家族的保守结构域。二级结构中α-螺旋(alpha helix)占37.21%、β-延伸(β-extended)占23.25%、无规则卷曲(random coil)占39.54%。同源性分析显示,sbCHI基冈与黄芩(ADQ13184.1)核苷酸序列相似性为99.69%、氨基酸序列相似性为99.07%,仅在第31、160位不同。结论成功从黄芩愈伤组织中克隆得到sbCHI,为进一步鉴定sbCHI基因的功能及黄芩苷的合成生物学研究提供基础。
Objective To clone the chalcone isomerase (CHI) gene from Scutellaria baicalensis callus and analyze its bioinformatics. Methods RNA was extracted from Scutellaria baicalensis callus and transcribed into cDNA. The specific primers were designed and the sbCHI gene of Scutellaria baicalensis Georgi was cloned. By bioinformatics analysis of the characteristics of the gene protein, the use of MEGA5.1 sbCHI and related species chalcone isomerase gene phylogenetic tree. Results The sbCHI gene (GeneBank accession number KP064512) was 648 bp in length and contained a complete open reading frame encoding 215 amino acids. As unstable hydrophilic protein, the molecular weight of sbCHI encoded protein was 22 980 and the isoelectric point (pI) was 5.09. It contained no signal peptide, no transmembrane region and had a conserved domain of chalcone superfamily. In the secondary structure, α helix accounted for 37.21%, β-extended accounted for 23.25%, random coil accounted for 39.54%. Homology analysis showed that the nucleotide sequence similarity of sbCHI Kei Gang and Scutellaria baicalensis (ADQ13184.1) was 99.69%, and the amino acid sequence similarity was 99.07%, only different at the 31st and the 160th. Conclusion sbCHI was successfully cloned from callus of Scutellaria baicalensis Georgi, which provided a basis for further studies on the function of sbCHI gene and the synthetic biology of baicalin.