芍药肌动蛋白基因组DNA的克隆及分析

Cloning and analysis of Actin from Paeonia lactiflora

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 Genomic gene encoding Actin in Paeonia lactiflora was cloned in order to clarify the gene organization and its expression levels in different tissues in herbaceous peony. Methods Based on cDNA sequences of Actin genes isolated from P. lactiflora reported by our laboratory, one pair of PCR primers was designed. PCR products ofActin genomic gene were successfully amplified with total genomic DNA extracted from herbecous peony cv. ＂Taohuafeixue＂ as template by high-fidelity DNA polymerase KOD-Plus, and then they were cloned to pMD 18-T vector and be sequenced. The exons and introns ofActin gene were predicted with bioinformatic sol, wares. The homology was analyzed by Blastn at the nucleotide level and the molecular phylogenetic tree was constructed with MEGA5.0 software. Based on sequencing results, one pair of PCR primers was designed, and the expression levels of Actin gene in the roots, stems, flowers, and leaves in herbaceous peony were semi-quantified. Results The sequencing results showed that Actin gene of herbaceous peony was 1 405 bp length, which contained four exons and three introns. All the splicing sites in three introns were conservative at 5＇ donor with GU and 3＇ receptor with AG. It encoded 377 amino acids, and GenBank accession number was KF363830. A pair of PCR primers was designed and one of them was spaned the first intron, which could effectively prevent the false postive by genomic DNA contamination. The semi-quantitative RT-PCR analysis showed that the expression levels ofActin gene in the roots, stems, leaves, and flowers of herbaceous peony were almost constant. Conclusion It is the first report on cloning and gene organization clarification of genomic gene encoding Actin in P. lactiflora. The semi-quantitative analyses indicate that Actin gene can be used as the internal standard gene for the expression analysis of the functional genes in herbaceous peony.