摘要
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 Cloning of Actin gene from Paeonia lactiflora and expression analysis of Actin gene with RT-PCR technique. Methods Based on cDNA sequences of Actin genes isolated from the related species reported in GenBank, one pair of PCR primers was designed. PCR products of Actin gene were successfully amplified with RT-PCR technique using cDNA synthesized from the total RNA extracted from the roots ofP. lactiflora ＇Taohuafeixue＇ as template, and then they were cloned to pMD18-T vector with TA cloning method. The positive clones which were characterized with colony PCR, plasmid PCR, and enzyme digestion method were sequenced. Based on the sequencing results, one pair of PCR primers was designed, and the expression profile of Actin gene in the roots, stems, flowers, and leaves in P. lactiflora were semi-quantified with RT-PCR technique. Results The sequencing results showed that the length ofActin gene ofP lactiflora was 1 134 bp, encoding 377 amino acids, whose GenBank accession number was JX310002. Sequence analysis showed that Actin ofP. lactiflora contained three kinds of characteristic signal sequences, whose homologous similarity in amino acid level with other plants was up to 99%. Based on homology modeling analysis, it revealed that there were four domains in its 3D structure. Bioinformatic software predicted that the molecular weight of Actin of herbaceous peony was 4.17 × 104, and the i soelectric point （pI） was 5.31. It was a hydrophilic and stable protein which was located in cytoplasm, without the transmembrane domain or signal peptide. Semi-quantitative RT-PCR analysis showed that the gene expression levels of Actin gene in the roots, stems, flowers, and leaves of P. lactiflora were almost constant. Conelusion It is the first report on the cloning of Actin gene from P lactiflora and the semi-quantitative analysis indicates that Actin gene can be used as the internal standard gene for the expression analysis of functional genes in P lactiflora. This project plays a base for the effective applic
出处
《中草药》
CAS
CSCD
北大核心
2013年第15期2136-2142,共7页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(U1204323)
河南省高等学校青年骨干教师资助计划(2010GGJS-075)
河南省科技厅国际合作项目资助(134300510052)
