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目的:克隆冬凌草乙酰辅酶A酰基转移酶(acetyl-CoA C-acetyltransferase,AACT)基因并分析其在冬凌草不同组织中的表达模式。方法:在冬凌草转录组测序的基础上设计特异引物,采用逆转录PCR技术克隆冬凌草IrAACT基因全长,并进行生物信息学分析,采用实时荧光定量PCR法分析其组织表达模式。结果:克隆得到的IrAACT基因全长1 254 bp,编码417个氨基酸,该序列与丹参等植物中的AACT基因有较高的同源性。生物信息学预测IrAACT具有Ⅱ型硫解酶催化作用的活性中心结构域。实时荧光定量PCR表明,IrAACT在冬凌草花和叶中的表达量明显高于根、茎和愈伤组织。结论:该研究获得了IrAACT基因的全长cDNA序列,并揭示了其在不同组织中的表达差异,为进一步阐述该基因在冬凌草二萜类成分合成途径中的功能奠定了基础。
OBJECTIVE: To clone the gene of acetyl-CoA-acetyltransferase (AACT) from Rubia cordyceps and analyze its expression pattern in different tissues of Rubia cordata. Methods: Specific primers were designed based on the sequence analysis of Rubia cordyceps transcriptome. The full length of IrAACT gene was cloned by reverse transcription polymerase chain reaction (RT - PCR) and analyzed by bioinformatics method. The expression pattern of the gene was analyzed by real - time fluorescence quantitative PCR. Results: The cloned IrAACT gene was 1 254 bp in length and encoded a polypeptide of 417 amino acids. This gene has high homology with AACT gene in plants such as Salvia miltiorrhiza. Bioinformatics predicts that IrAACT has an active center domain that catalyzes the type II thiolase activity. Real-time fluorescence quantitative PCR showed that the expression of IrAACT in the flowers and leaves of Rubestein was significantly higher than that of roots, stems and callus. Conclusion: The full-length cDNA sequence of IrAACT gene was obtained in this study and the differences in expression of IrAACT gene were revealed in different tissues, which laid the foundation for further elucidation of the function of this gene in the synthesis pathway of diterpenoid constituents.