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[摘要]
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基因与黄芩(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。
[Key word]
[Abstract]
Objective To clone chalcone isomerase (sbCHI) gene from the callus of Scutellaria baicalensis and to analyze its bioinformatics. Methods RNA was obtained from scutellariae callus, cDNA was reversely transcribed, specific primers were designed, and then CHI was cloned. The protein characteristics was analyzed using bioinformatics and the phylogenetic tree of CHI was constructed using MEGA5.1. Results The 648 bp sbCHI (accession number: KP064512) sequence was obtained, which has a complete open reading frame (ORF), encoding an unstable protein with 215 amino acids. The sbCHI encoding protein has isoelectric point (pI) of 5.09 and a calculated molecular weight about 22 980, without transmembrane regions and signal peptide has a conserved domain of chalcone-flavanone isomerase family. In the secondary structure, the percentage of alpha helix, β-extended , and random coil were 37.21%, 23.25%, and 39.54%, respectively. The homologous analysis indicates the nucleotide sequence 99.69% similarity and the amino acid sequence 99.07% similarity with S. baicalensis (ADQ13184.1), only in 31 and 160 were different. Conclusion The sbCHI in scutellariae callus is successfully cloned, which provides the foundation for further characterization sbCHI functionality and the synthetic biology research of baicalin.
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[基金项目]
国家自然科学基金资助项目(31071292);国家科技支撑计划项目(2011BAI0301-02);吉林省科技成果转化项目(20125068)