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目的克隆芍药肌动蛋白(Actin)基因并应用RT-PCR技术分析Actin基因在芍药不同组织中的表达情况。方法根据近缘物种Actin基因的保守序列设计一对PCR扩增引物,以“桃花飞雪”芍药根部总RNA反转录而成的cDNA为模板,采用RT-PCR的方法扩增出芍药Actin cDNA全长并克隆至pMD18-T载体上,阳性克隆经菌落PCR、质粒PCR及酶切鉴定后进行测序。基于测序结果设计特异性PCR引物,应用RT-PCR技术分析Actin基因在芍药根、茎、花、叶不同组织中的表达情况。结果测序结果表明芍药Actin基因全长1 134 bp序列,共编码377个氨基酸,GenBank登录号为JX310002;序列分析表明芍药Actin蛋白中存在3种肌动蛋白特征信号序列,与其他植物肌动蛋白氨基酸同源性达99%;基于同源模建预测的3D结构中含有4个结构域;生物信息学软件预测芍药肌动蛋白的相对分子质量为4.17×104,等电点(pI)为5.31,是一个定位于胞液、不含跨膜域、不含信号肽分子的亲水性、稳定蛋白;半定量RT-PCR技术分析表明Actin基因在芍药根、茎、叶、花组织中的表达量保持恒定。结论首次从芍药中克隆了Actin基因,半定量RT-PCR表达分析结果表明Actin基因适合作为芍药功能基因表达分析的内标基因,为有效利用该基因奠定了基础。
Objective To clone Actin gene of Paeonia lactiflora and analyze the expression of Actin gene in different tissues of Paeonia lactiflora by RT-PCR. Methods A pair of PCR amplification primers was designed according to the conserved sequence of Actin gene from relative species. The reverse transcription of total RNA of Peach Blossom and Peony root was used as template to amplify peony Actin cDNA was cloned into pMD18-T vector. The positive clones were sequenced after colony PCR, plasmid PCR and restriction enzyme digestion. Specific PCR primers were designed based on sequencing results, and the expression of Actin gene in different tissues of root, stem, flower and leaf of Paeonia lactiflora was analyzed by RT-PCR. Results The sequencing results showed that Actin gene was 1 134 bp in length and encoded a total of 377 amino acids. GenBank accession number was JX310002. The sequence analysis indicated that there are three actin signal sequences in Actin of Paeonia lactiflora, which interacted with other plant actin amino acids And the homology was 99%. The 3D structure based on homology modeling contained four domains. The bioinformatics software predicted that the relative molecular mass of peony actin was 4.17 × 104, the isoelectric point (pI) was 5.31, Is a hydrophilic and stable protein localized in cytosol, transmembrane domain and free of signal peptide. Semi-quantitative RT-PCR analysis showed that the expression of Actin gene in root, stem, leaf and flower of Paeonia keep constant. Conclusion The Actin gene was cloned from Paeonia lactiflora for the first time. The results of semi-quantitative RT-PCR analysis indicated that Actin gene was suitable as an internal standard gene for gene expression analysis of Paeonia lactiflora, which laid the foundation for the effective use of this gene.