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目的克隆芍药Paeonia lactiflora肌动蛋白(Actin)基因组DNA序列并解析基因结构,分析Actin基因在芍药不同组织中的表达情况。方法根据本课题组报道的芍药Actin基因cDNA序列(JX310002)设计特异性引物,以芍药栽培品种“桃花飞雪”总DNA为模板,用KOD-Plus高保真DNA聚合酶扩增芍药Actin基因组基因,克隆PCR产物并进行测序。应用生物信息学软件预测芍药Actin基因的外显子及内含子,基于Blastn程序分析芍药Actin基因在核苷酸水平上的同源性,应用MEGA5.0软件构建分子系统进化树;设计跨越内含子的半定量RT-PCR扩增引物,分析芍药Actin基因在芍药根、茎、叶、花中的表达情况。结果测序结果表明芍药Actin基因组DNA序列全长1 405 bp,含4个外显子和3个内含子,3个内含子中共6个剪接位点均遵循高等真核生物5’端供位GU与3’端受位AG模式;共编码377个氨基酸,GenBank登录号为KF363830。设计了一对半定量RT-PCR扩增引物,其中上游引物跨越了芍药Actin基因的第1个内含子,可有效防止由DNA污染而造成RT-PCR扩增的假阳性;半定量RT-PCR分析结果表明Actin基因在芍药根、茎、叶、花等不同组织中的表达量保持恒定。结论首次克隆了芍药Actin基因组DNA序列并明确了其基因结构,半定量RT-PCR分析结果表明Actin基因可以作为芍药功能基因表达分析的内标基因。
Objective To clone the Actin genomic DNA of Paeonia lactiflora and analyze the gene structure and analyze the expression of Actin gene in different tissues of Paeonia lactiflora. Methods Based on the Actin gene cDNA sequence (JX310002) reported by our group, we designed specific primers to amplify the Actin gene of Paeonia lactiflora with KOD-Plus high-fidelity DNA polymerase using the total DNA of peony cultivars “Peach Blossom” as template Genes were cloned and sequenced. The bioinformatics software was used to predict the exon and intron of Actin gene of Paeonia lactiflora. Based on the Blastn program, the nucleotide homology of Actin gene was determined. The phylogenetic tree was constructed using MEGA5.0 software. The semi-quantitative RT-PCR primers were used to analyze the expression of Actin gene from Paeonia osidei, root, stem, leaf and flower of Paeonia lactiflora. Results The sequencing results showed that the full-length cDNA sequence of Actinomyces peony was 1 405 bp in length, containing 4 exons and 3 introns. The 6 splice sites of 3 introns all followed the 5 ’end of higher eukaryotes GU and 3 ’end are in AG mode; a total of 377 amino acids are encoded, GenBank accession number is KF363830. A pair of semi-quantitative RT-PCR amplification primers was designed, in which the upstream primer spanned the first intron of Actin gene of Paeonia lactiflora, which could effectively prevent the false positive of RT-PCR amplification caused by DNA contamination. Semi-quantitative RT- PCR analysis showed that the expression of Actin gene was constant in different tissues of root, stem, leaf, flower and other tissues of Paeonia lactiflora. Conclusions The Actin gene sequence of Paeonia lactiflora was cloned for the first time and its gene structure was clarified. The result of semi-quantitative RT-PCR showed that Actin gene could be used as the internal standard gene of Paeonia lactiflora functional gene expression analysis.